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1
McKinnon, Thomas A. J., Agata Anna Nowak, Alina Hua, Carolyn Millar, and Michael Laffan. "Flow Analysis of Von Willebrand Factor Collagen Binding Mutants." Blood 118, no. 21 (November 18, 2011): 2213. http://dx.doi.org/10.1182/blood.v118.21.2213.2213.
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Abstract Abstract 2213 Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress. Disclosures: No relevant conflicts of interest to declare.
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Shida, Yasuaki, Christine Brown, Jeff Mewburn, Kate Sponagle, Ozge Danisment, Barbara Vidal, Carol A. Heagadorn, and David Lillicrap. "Comprehensive In Vitro and In Vivo Characterization of Loss and Gain-of-Function Von Willebrand Factor Collagen Binding Variants Using a Mouse Model System,." Blood 118, no. 21 (November 18, 2011): 3266. http://dx.doi.org/10.1182/blood.v118.21.3266.3266.
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Abstract Abstract 3266 Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of injury. Rare mutations in the VWF A3 domain, that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been relatively limited. Thus, in this study, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 and A1 domains using comprehensive in vitro and in vivo approaches. All of the collagen binding variant AAs are conserved in mice. 6 loss-of-function (S1731T, W1745C, S1783A, H1786D, A1 deletion, A3 deletion) and 1 gain-of-function (L1757A) variant was generated in the context of the mouse VWF cDNA. The 4 loss-of-function missense mutants have all been described in patients with mild bleeding phenotypes. The recombinant mouse VWFs (rmVWF) were synthesized in HEK293T cells and analyzed for type I and III collagen binding in both a static assay (CBA) and a flow-based assay at 2,500s−1 in which VWF is bound to collagen on a surface, and labeled platelet adhesion is quantified. The multimer profile of all the rmVWFs was normal. The expression level of the rmVWF derived from HEK293T cells was quantified. W1745C and the A3 deletion showed significantly lower levels of expression and the A1 deletion mutant showed strong intracellular retention. In the static collagen binding assay, S1731T showed almost normal binding to collagen type I and a 50% reduction in binding to collagen type III. The other 3 missense variants, W1745C, S1783A and H1786D, showed reduced binding to both collagens I and III, and the A3 deletion mutant showed absent binding. In the in vitro flow assay, the sensitivity to detect defects in collagen binding was superior to the static assay, although the patterns of binding defects were similar. W1745C showed similar low levels of platelet adhesion to both types of collagen, while S1783A and H1786D showed a lack of platelet binding on the collagen III surface similar to the A3 deletion mutant, and a reduced binding to collagen type I similar to W1745C. The gain-of-function mutant showed consistent enhanced collagen binding and platelet adhesion in the static and flow assays, respectively. In vivo studies delivered the mVWF cDNAs with a strong liver specific promoter by hydrodynamic injection. At 7 days post-delivery, the VWF:Ag levels in the WT and collagen binding variant mice were similar, apart from the W1745C mutant, that showed 14.6% levels compared to WT. Platelet counts and multimer patterns were normal with the collagen binding variants. In vivo intravital microscopy studies were performed using the cremaster arteriolar model when VWF levels were in a physiological range. Thrombosis was induced by 10%FeCl3 applied for 3 mins. Platelets were labeled in vivo by Rhodamine 6G and the thrombus development was analyzed by spinning disc confocal microscopy. Loss-of-function mutants showed transient platelet adhesion at the site of injury, however the adhesion was unstable and vessel occlusion was not observed. Using three complementary experimental systems we have been able to confirm the collagen binding defects in this group of variant VWFs. There is a differential sensitivity to the two forms of collagen and of the three experimental systems. The A3 deletion mutant consistently resulted in the most severe phenotype while the missense mutants showed variable degrees of functional deficit. Disclosures: No relevant conflicts of interest to declare.
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Shitomi, Yasuyuki, Ida B. Thøgersen, Noriko Ito, Birgit Leitinger, Jan J. Enghild, and Yoshifumi Itoh. "ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)." Molecular Biology of the Cell 26, no. 4 (February 15, 2015): 659–73. http://dx.doi.org/10.1091/mbc.e14-10-1463.
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Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1–dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1-mediated cell adhesion to achieve efficient cell migration on collagen matrices.
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Petersen, F. C., S. Assev, H. C. van der Mei, H. J. Busscher, and A. A. Scheie. "Functional Variation of the Antigen I/II Surface Protein in Streptococcus mutans and Streptococcus intermedius." Infection and Immunity 70, no. 1 (January 2002): 249–56. http://dx.doi.org/10.1128/iai.70.1.249-256.2002.
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ABSTRACT Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.
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Guimarães-Souza, Nadia Karina, Liliya Marsovna Yamaleyeva, Baisong Lu, Ana Claudia Mallet de Souza Ramos, Colin Edward Bishop, and Karl Erik Andersson. "Superoxide overproduction and kidney fibrosis: a new animal model." Einstein (São Paulo) 13, no. 1 (March 2015): 79–88. http://dx.doi.org/10.1590/s1679-45082015ao3179.
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Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis.
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van der Plas, R., Lucio Gomes, J. Marquart, Tom Vink, Joost C. Meijers, Philip de Groot, Jan Sixma, and Eric Huizinga. "Binding of von Willebrand Factor to Collagen Type III: Role of Specific Amino Acids in the Collagen Binding Domain of vWF and Effects of Neighboring Domains." Thrombosis and Haemostasis 84, no. 12 (2000): 1005–11. http://dx.doi.org/10.1055/s-0037-1614163.
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SummaryBinding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the threedimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain.In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD, indicating that the A2 domain somehow modulates binding of vWF to collagen type III.Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.
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Marx, Isabelle, Olivier D. Christophe, Peter J. Lenting, Alain Rupin, Marie-Odile Vallez, Tony J. Verbeuren, and Cécile V. Denis. "Altered thrombus formation in von Willebrand factor–deficient mice expressing von Willebrand factor variants with defective binding to collagen or GPIIbIIIa." Blood 112, no. 3 (August 1, 2008): 603–9. http://dx.doi.org/10.1182/blood-2008-02-142943.
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Abstract The role of von Willebrand factor (VWF) in thrombosis involves its binding to a number of ligands. To investigate the relative importance of these particular interactions in the thrombosis process, we have introduced mutations into murine VWF (mVWF) cDNA inhibiting VWF binding to glycoprotein (Gp) Ib, GPIIbIIIa, or to fibrillar collagen. These VWF mutants were expressed in VWF-deficient mice (VWF−/−) by using an hydrodynamic injection approach, and the mice were studied in the ferric chloride–induced injury model. Expression of the collagen and the GPIIbIIIa VWF-binding mutants in VWF−/− mice resulted in delayed thrombus growth and significantly increased vessel occlusion times compared with mice expressing wild-type (WT) mVWF (30 ± 3 minutes and 38 ± 4 minutes for the collagen and GPIIbIIIa mutants, respectively, vs 19 ± 3 minutes for WT mVWF). Interestingly, these mutants were able to correct bleeding time as efficiently as WT mVWF. In contrast, VWF−/− mice expressing the GPIb binding mutant failed to restore thrombus formation and were bleeding for as long as they were observed, confirming the critical importance of the VWF-GPIb interaction. Our observations suggest that targeting the VWF-collagen or VWF-GPIIbIIIa interactions could be an interesting alternative for new antithrombotic strategies.
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Cruz, Miguel A., and Liza D. Morales. "Mutations in the von Willebrand Factor A1 Domain That Increase the Affinity for Collagen Have Different Effects on the Binding to Glycoprotein Ib." Blood 104, no. 11 (November 16, 2004): 3661. http://dx.doi.org/10.1182/blood.v104.11.3661.3661.
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Abstract The interaction of plasma von Willebrand factor (VWF) with collagen at the site of vascular injury plays a critical role in the initiation of thrombus formation under high shear stress. It does this by forming a bridge between the fibrils of collagen in the subendothelium and the platelet glycoprotein Ib-IX-V complex (GPIb). The A1 domain of VWF is the binding site for GPIb whereas the collagen-function of VWF is controlled by both A1 and A3 domains. The VWF-A3 domain is important to support binding to fibrils of collagen Types I and III while the A1 domain is involved in the binding to microfibrillar collagen Type VI. It is assumed that the interaction of VWF with fibrillar collagen (via the A3 domain) may regulate the expression of the GPIb-binding site in the A1 domain. However, there is no a definite data to substantiate that hypothesis. Our goal was to demonstrate that a direct interaction between the A1 domain and fibrillar collagen Types I or III exposes the GPIb binding site. Thus, we postulated that platelet GPIb is able to interact with isolated A1 domain that is bound to collagen. We have demonstrated that the VWF-A1 protein binds specifically to human placenta collagen Types I and III with a KD ~ 200 nM by using surface plasmon resonance (SPR). Using plasma-free blood, we have provided strong evidence that isolated VWF-A1 domain bound to either collagen Type I or III is able to support platelet adhesion under high flow conditions. This platelet interaction was effectively blocked with antibodies against either GPIb or A1 domain. These results clearly show the ability of the A1 domain to concurrently interact with both GPIb and collagen fibrils and they also suggest that the collagen-A1 binding may regulate the expression of the GPIb-binding site in the A1 domain. To test this hypothesis, we analyzed three residues that in a previous mutagenesis study they increased the binding of VWF to GPIb, reasoning that they may have an effect on the collagen binding activity as well. The three residues are located in the a7 helix (rear face) of the folded A1 domain and mutagenesis studies of other I(A)-domains have demonstrated that this helix plays a role in regulating the affinity of the ligand-binding. We introduced point mutations into the 3 residues and the recombinant mutant proteins were expressed in bacteria. The three mutants (R687E, D688R, and E689R) were purified as wild type and their structural integrity was confirmed with three conformation-specific antibodies. All the mutants bound to both collagens Type I or III with an affinity much higher than the wild type (WT) (KD~ 9 -1 nM). The mutants were assessed by their ability to mediate platelet adhesion to collagen, and their ability to inhibit both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. Interestingly, in the three assays the R687E mutant had an activity higher than WT while the D688R had a markedly decrease activity. The mutant E689R had an activity similar to that of WT for the three assays. Together our data indicate that a direct association between the VWF-A1 domain and collagen fibrils influences the expression of GPIb binding function in VWF. Further, these data indicate that residue R687 located in the a7 helix plays a novel and important role in modulating the collagen/A1/GPIb binding.
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Mintz, Keith P. "Identification of an extracellular matrix protein adhesin, EmaA, which mediates the adhesion of Actinobacillus actinomycetemcomitans to collagen." Microbiology 150, no. 8 (August 1, 2004): 2677–88. http://dx.doi.org/10.1099/mic.0.27110-0.
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Actinobacillus actinomycetemcomitans is an aetiologic agent in the development of periodontal and some systemic diseases in humans. This pathogen localizes to the underlying connective tissue of the oral cavity in individuals with periodontal disease. The adhesion of A. actinomycetemcomitans to extracellular matrix components of the connective tissue prompted this study to identify gene products mediating the interaction of A. actinomycetemcomitans to these molecules. A transposon mutagenesis system was optimized for use in A. actinomycetemcomitans and used to generate an insertional mutant library. A total of 2300 individual insertion transposon mutants were screened for changes in the adhesion to collagen and fibronectin. Mutants were identified which exhibited the following phenotypes: a decrease in collagen binding; a decrease in fibronectin binding; a decrease in binding to both proteins; and an increase in binding to both collagen and fibronectin. The identification of mutants defective in adhesion to the individual proteins indicates that distinct adhesins are expressed by this organism. Molecular analysis of these mutants implicated 11 independent loci in protein adhesion. One gene, emaA, is likely to encode a direct mediator of collagen adhesion, based on predicted protein features homologous to the collagen-binding protein YadA of Yersinia enterocolitica. EmaA was localized to the outer membrane, as expected for an adhesin. Reduction in fibronectin adhesion appeared to be influenced by abrogation of proteins involved in molybdenum-cofactor biosynthesis. Several other loci identified as reducing or increasing adhesion to both collagen and fibronectin are suggested to be involved in regulatory cascades that promote or repress expression of collagen and fibronectin adhesins. Collectively, the results support the hypothesis that A. actinomycetemcomitans host colonization involves afimbrial adhesins for extracellular matrix proteins, and that the expression of adhesion is modulated by global regulatory mechanisms.
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Lankhof, Hanneke, Conchi Damas, Marion Schiphorst, Martin IJsseldijk, Madelon Bracke, Miha Furlan, Philip de Groot, Tom Vink, and Jan Sixma. "Recombinant vWF Type 2A Mutants R834Q and R834W Show a Defect in Mediating Platelet Adhesion to Collagen, Independent of Enhanced Sensitivity to a Plasma Protease." Thrombosis and Haemostasis 81, no. 06 (1999): 976–83. http://dx.doi.org/10.1055/s-0037-1614609.
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SummaryType 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (vWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 μg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis.
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Mesbahi, Hiva, Kim B. Pho, Andrea J. Tench, Victoria L. Leon Guerrero, and Lesley T. MacNeil. "Cuticle Collagen Expression Is Regulated in Response to Environmental Stimuli by the GATA Transcription Factor ELT-3 in Caenorhabditis elegans." Genetics 215, no. 2 (March 30, 2020): 483–95. http://dx.doi.org/10.1534/genetics.120.303125.
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The nematode Caenorhabditis elegans is protected from the environment by the cuticle, an extracellular collagen-based matrix that encloses the animal. Over 170 cuticular collagens are predicted in the C. elegans genome, but the role of each individual collagen is unclear. Stage-specific specialization of the cuticle explains the need for some collagens; however, the large number of collagens suggests that specialization of the cuticle may also occur in response to other environmental triggers. Missense mutations in many collagen genes can disrupt cuticle morphology, producing a helically twisted body causing the animal to move in a stereotypical pattern described as rolling. We find that environmental factors, including diet, early developmental arrest, and population density can differentially influence the penetrance of rolling in these mutants. These effects are in part due to changes in collagen gene expression that are mediated by the GATA family transcription factor ELT-3. We propose a model by which ELT-3 regulates collagen gene expression in response to environmental stimuli to promote the assembly of a cuticle specialized to a given environment.
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Sebghati, Tricia A., and Steven Clegg. "Construction and Characterization of Mutations within theKlebsiella mrkD1P Gene That Affect Binding to Collagen Type V." Infection and Immunity 67, no. 4 (April 1, 1999): 1672–76. http://dx.doi.org/10.1128/iai.67.4.1672-1676.1999.
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ABSTRACT The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within themrkD 1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD 1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1Pprotein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.
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Bullard, D. C., L. A. Hurley, I. Lorenzo, L. M. Sly, A. L. Beaudet, and N. D. Staite. "Reduced susceptibility to collagen-induced arthritis in mice deficient in intercellular adhesion molecule-1." Journal of Immunology 157, no. 7 (October 1, 1996): 3153–58. http://dx.doi.org/10.4049/jimmunol.157.7.3153.
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Abstract Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the firm adhesion of leukocytes to venular endothelium and facilitates leukocyte extravasation from the vasculature into inflamed tissue. In addition, ICAM-1 is an important costimulatory molecule during Ag presentation to lymphocytes. Using mice deficient in ICAM-1, we have investigated the role of this molecule in the development of collagen-induced arthritis. After immunization with type II collagen, 71% of wild-type mice developed arthritis compared with 50% of ICAM-1 heterozygote mutants and 18% of ICAM-1 homozygous mutants. In those ICAM-1 mutants that developed arthritis, the mean day of onset, the mean number of involved paws, and the severity of paw inflammation were not significantly different from those in wild-type mice. The reduced incidence of arthritis in the ICAM-1 homozygous mutant mice was not due to lack of immunity to type II collagen, since these mice developed similar levels of anti-type II collagen IgG compared with wild-type mice and had a positive delayed-type hypersensitivity reaction to type II collagen. The reduction of arthritis in heterozygous as well as homozygous deficient mice indicates that expression of ICAM-1 can be a pivotal variable in the pathogenesis of collagen-induced arthritis in mice. The results suggest that naturally occurring genetic variation in the expression of ICAM-1 or related inflammatory cell adhesion molecules might influence susceptibility to the complex disease of rheumatoid arthritis in humans and support the concept that pharmacologic approaches to chronic reduction in the expression or the function of ICAM-1 may be of therapeutic value.
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Lawrence, Elizabeth A., Erika Kague, Jessye A. Aggleton, Robert L. Harniman, Karen A. Roddy, and Chrissy L. Hammond. "The mechanical impact of col11a2 loss on joints; col11a2 mutant zebrafish show changes to joint development and function, which leads to early-onset osteoarthritis." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1759 (September 24, 2018): 20170335. http://dx.doi.org/10.1098/rstb.2017.0335.
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Collagen is the major structural component of cartilage, and mutations in the genes encoding type XI collagen are associated with severe skeletal dysplasias (fibrochondrogenesis and Stickler syndrome) and early-onset osteoarthritis (OA). The impact of the lack of type XI collagen on cell behaviour and mechanical performance during skeleton development is unknown. We studied a zebrafish mutant for col11a2 and evaluated cartilage, bone development and mechanical properties to address this. We show that in col11a2 mutants, type II collagen is made but is prematurely degraded in maturing cartilage and ectopically expressed in the joint. These changes are correlated with increased stiffness of both bone and cartilage; quantified using atomic force microscopy. In the mutants, the skeletal rudiment terminal region in the jaw joint is broader and the interzone smaller. These differences in shape and material properties impact on joint function and mechanical performance, which we modelled using finite element analyses. Finally, we show that col11a2 heterozygous carriers reach adulthood but show signs of severe early-onset OA. Taken together, our data demonstrate a key role for type XI collagen in maintaining the properties of cartilage matrix; which when lost leads to alterations to cell behaviour that give rise to joint pathologies. This article is part of the Theo Murphy meeting issue ‘Mechanics of development’.
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Yu, Chunxiao, Teresa Ruiz, Christopher Lenox, and Keith P. Mintz. "Functional Mapping of an Oligomeric Autotransporter Adhesin of Aggregatibacter actinomycetemcomitans." Journal of Bacteriology 190, no. 9 (February 29, 2008): 3098–109. http://dx.doi.org/10.1128/jb.01709-07.
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ABSTRACT Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.
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Hill, Katherine L., Brian D. Harfe, Carey A. Dobbins, and Steven W. L'Hernault. "dpy-18 Encodes an α-Subunit of Prolyl-4-Hydroxylase in Caenorhabditis elegans." Genetics 155, no. 3 (July 1, 2000): 1139–48. http://dx.doi.org/10.1093/genetics/155.3.1139.
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Abstract Collagen is an extracellular matrix (ECM) component encoded by a large multigene family in multicellular animals. Procollagen is post-translationally modified by prolyl-4-hydroxylase (EC 1.14.11.2) before secretion and participation in ECM formation. Therefore, collagen processing and regulation can be studied by examining this required interaction of prolyl-4-hydroxylase with procollagen. High-resolution polymorphism mapping was used to place the Caenorhabditis elegans dpy-18 gene on the physical map, and we show that it encodes a prolyl-4-hydroxylase α catalytic subunit. The Dpy phenotype of dpy-18(e364) amber mutants is more severe when this mutation is in trans to the noncomplementing deficiency tDf7, while the dpy-18(e499) deletion mutant exhibits the same phenotype as dpy-18(e499)/tDf7. Furthermore, dpy-18 RNA interference (RNAi) in wild-type worms results in Dpy progeny, while dpy-18 (RNAi) in dpy-18(e499) mutants does not alter the Dpy phenotype of their progeny. These observations suggest that the dpy-18 null phenotype is Dpy. A dpy-18::gfp promoter fusion construct is expressed throughout the hypodermis within the cells that abundantly produce the cuticle collagens, as well as in certain head and posterior neurons. While prolyl-4-hydroxylase has been studied extensively by biochemical techniques, this is the first report of a mutationally defined prolyl-4-hydroxylase in any animal.
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Lankhof, Hanneke L., Maggy van Hoeij, Marion E. Schiphorst, Madelon Bracke, Ya-Ping Wu, Martin J. W. Ijsseldijk, Tom Vink, Philip G. de Groot, and Jan J. Sixma. "A3 Domain Is Essential for Interaction of von Willebrand Factor with Collagen Type III." Thrombosis and Haemostasis 75, no. 06 (1996): 950–58. http://dx.doi.org/10.1055/s-0038-1650400.
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Summaryvon Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; ΔA1-vWF; Sixma et al. Eur. J. Biochem. 196,369,1991 [1]) or the A3 domain (residues 910-1113; ΔA3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for ΔA3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) ΔAl-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of ΔA3-vWF to collagen type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-ΔA3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, ΔA3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas ΔAl-vWF did. The binding of 125I-ΔA3-vWF could be blocked by excess unlabeled vWF but not by ΔA1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.
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Chakravarti, Shukti, Terry Magnuson, Jonathan H. Lass, Karl J. Jepsen, Christian LaMantia, and Heidi Carroll. "Lumican Regulates Collagen Fibril Assembly: Skin Fragility and Corneal Opacity in the Absence of Lumican." Journal of Cell Biology 141, no. 5 (June 1, 1998): 1277–86. http://dx.doi.org/10.1083/jcb.141.5.1277.
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Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.
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Tanabe, Tomoya, Miharu Maeda, Kota Saito, and Toshiaki Katada. "Dual function of cTAGE5 in collagen export from the endoplasmic reticulum." Molecular Biology of the Cell 27, no. 13 (July 2016): 2008–13. http://dx.doi.org/10.1091/mbc.e16-03-0180.
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Two independent functions of cTAGE5 have been reported in collagen VII export from the endoplasmic reticulum (ER). cTAGE5 not only forms a cargo receptor complex with TANGO1, but it also acts as a scaffold to recruit Sec12, a guanine-nucleotide exchange factor for Sar1 GTPase, to ER exit sites. However, the relationship between the two functions remains unclear. Here we isolated point mutants of cTAGE5 that lost Sec12-binding ability but retained binding to TANGO1. Although expression of the mutant alone could not rescue the defects in collagen VII secretion mediated by cTAGE5 knockdown, coexpression with Sar1, but not with the GTPase-deficient mutant, recovered secretion. The expression of Sar1 alone failed to rescue collagen secretion in cTAGE5-depleted cells. Taken together, these results suggest that two functionally irreplaceable and molecularly separable modules in cTAGE5 are both required for collagen VII export from the ER. The recruitment of Sec12 by cTAGE5 contributes to efficient activation of Sar1 in the vicinity of ER exit sites. In addition, the GTPase cycle of Sar1 appears to be responsible for collagen VII exit from the ER.
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Topf, Ulrike, and Ruth Chiquet-Ehrismann. "Genetic interaction between Caenorhabditis elegans teneurin ten-1 and prolyl 4-hydroxylase phy-1 and their function in collagen IV–mediated basement membrane integrity during late elongation of the embryo." Molecular Biology of the Cell 22, no. 18 (September 15, 2011): 3331–43. http://dx.doi.org/10.1091/mbc.e10-10-0853.
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Teneurins are a family of phylogenetically conserved proteins implicated in pattern formation and morphogenesis. The sole orthologue in Caenorhabditis elegans, ten-1, is important for hypodermal cell migration, neuronal migration, path finding and fasciculation, gonad development, and basement membrane integrity of some tissues. However, the mechanisms of TEN-1 action remain to be elucidated. Using a genome-wide RNA interference approach, we identified phy-1 as a novel interaction partner of ten-1. phy-1 codes for the catalytic domain of collagen prolyl 4-hydroxylase. Loss of phy-1 significantly enhanced the embryonic lethality of ten-1 null mutants. Double-mutant embryos arrested during late elongation with epidermal defects, disruption of basement membranes, and detachment of body wall muscles. We found that deletion of phy-1 caused aggregation of collagen IV in body wall muscles in elongated embryos and triggered the loss of tissue integrity in ten-1 mutants. In addition, phy-1 and ten-1 each genetically interact with genes encoding collagen IV. These findings support a functional mechanism in which loss of ten-1, together with a reduction of assembled and secreted basement membrane collagen IV protein, leads to detachment of the epidermis from muscle cells during late elongation of the embryo when mechanical stress is generated by muscle contractions.
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Tang, Gaoyan, and Keith P. Mintz. "Glycosylation of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans Is Dependent upon the Lipopolysaccharide Biosynthetic Pathway." Journal of Bacteriology 192, no. 5 (January 8, 2010): 1395–404. http://dx.doi.org/10.1128/jb.01453-09.
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ABSTRACT The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.
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Gupta, Malini C., Patricia L. Graham, and James M. Kramer. "Characterization of α1(IV) Collagen Mutations in Caenorhabditis elegans and the Effects of α1 and α2(IV) Mutations on Type IV Collagen Distribution." Journal of Cell Biology 137, no. 5 (June 2, 1997): 1185–96. http://dx.doi.org/10.1083/jcb.137.5.1185.
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Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the α1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non–temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non–temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti–EMB-9 antisera and show intracellular accumulation of the α2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.
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Riddell, Anne, Keith Gomez, Carolyn Millar, G. Mellars, Simon A. Brown, Saher Gill, Michael Laffan, and Thomas A. J. McKinnon. "Characterisation of W1745C and S1783A, Two Novel Collagen Binding Defects in the A3 Domain of Von Willebrand Factor." Blood 112, no. 11 (November 16, 2008): 424. http://dx.doi.org/10.1182/blood.v112.11.424.424.
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Abstract Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs >0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.
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Kwan, Kin Ming, Michael K. M. Pang, Sheila Zhou, Soot Keng Cowan, Richard Y. C. Kong, Tim Pfordte, Bjorn R. Olsen, David O. Sillence, Patrick P. L. Tam, and Kathryn S. E. Cheah. "Abnormal Compartmentalization of Cartilage Matrix Components in Mice Lacking Collagen X: Implications for Function." Journal of Cell Biology 136, no. 2 (January 27, 1997): 459–71. http://dx.doi.org/10.1083/jcb.136.2.459.
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There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen α1(X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen α1(X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.
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Koseki, Kyohei, Aoi Yamamoto, Keisuke Tanimoto, Naho Okamoto, Fei Teng, Tomohiro Bito, Yukinori Yabuta, Tsuyoshi Kawano, and Fumio Watanabe. "Dityrosine Crosslinking of Collagen and Amyloid-β Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans." International Journal of Molecular Sciences 22, no. 23 (November 30, 2021): 12959. http://dx.doi.org/10.3390/ijms222312959.
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(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid β, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid β but increased the formation of dityrosine crosslinking in the amyloid β protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid β formation, which might promote its stabilization and toxic oligomerization.
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STEPLEWSKI, A., V. HINTZE, R. BRITTINGHAM, K. HOLMES, and A. FERTALA. "Conditional expression of collagen II mutants in SW1353 cells." Matrix Biology 25 (November 2006): S67. http://dx.doi.org/10.1016/j.matbio.2006.08.184.
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Hawkey-Noble, Alexia, Justin A. Pater, Roshni Kollipara, Meriel Fitzgerald, Alexandre S. Maekawa, Christopher S. Kovacs, Terry-Lynn Young, and Curtis R. French. "Mutation of foxl1 Results in Reduced Cartilage Markers in a Zebrafish Model of Otosclerosis." Genes 13, no. 7 (June 21, 2022): 1107. http://dx.doi.org/10.3390/genes13071107.
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Bone diseases such as otosclerosis (conductive hearing loss) and osteoporosis (low bone mineral density) can result from the abnormal expression of genes that regulate cartilage and bone development. The forkhead box transcription factor FOXL1 has been identified as the causative gene in a family with autosomal dominant otosclerosis and has been reported as a candidate gene in GWAS meta-analyses for osteoporosis. This potentially indicates a novel role for foxl1 in chondrogenesis, osteogenesis, and bone remodelling. We created a foxl1 mutant zebrafish strain as a model for otosclerosis and osteoporosis and examined jaw bones that are homologous to the mammalian middle ear bones, and mineralization of the axial skeleton. We demonstrate that foxl1 regulates the expression of collagen genes such as collagen type 1 alpha 1a and collagen type 11 alpha 2, and results in a delay in jawbone mineralization, while the axial skeleton remains unchanged. foxl1 may also act with other forkhead genes such as foxc1a, as loss of foxl1 in a foxc1a mutant background increases the severity of jaw calcification phenotypes when compared to each mutant alone. Our zebrafish model demonstrates atypical cartilage formation and mineralization in the zebrafish craniofacial skeleton in foxl1 mutants and demonstrates that aberrant collagen expression may underlie the development of otosclerosis.
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Swank, RT, M. Reddington, O. Howlett, and EK Novak. "Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha." Blood 78, no. 8 (October 15, 1991): 2036–44. http://dx.doi.org/10.1182/blood.v78.8.2036.2036.
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Abstract Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.
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Swank, RT, M. Reddington, O. Howlett, and EK Novak. "Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha." Blood 78, no. 8 (October 15, 1991): 2036–44. http://dx.doi.org/10.1182/blood.v78.8.2036.bloodjournal7882036.
Full textAbstract:
Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.
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Gistelinck, Charlotte, Ronald Y. Kwon, Fransiska Malfait, Sofie Symoens, Matthew P. Harris, Katrin Henke, Michael B. Hawkins, et al. "Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies." Proceedings of the National Academy of Sciences 115, no. 34 (August 6, 2018): E8037—E8046. http://dx.doi.org/10.1073/pnas.1722200115.
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The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.
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Tomita, Haruyoshi, and Yasuyoshi Ike. "Tissue-Specific Adherent Enterococcus faecalis Strains That Show Highly Efficient Adhesion to Human Bladder Carcinoma T24 Cells Also Adhere to Extracellular Matrix Proteins." Infection and Immunity 72, no. 10 (October 2004): 5877–85. http://dx.doi.org/10.1128/iai.72.10.5877-5885.2004.
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ABSTRACT The ability of Enterococcus faecalis clinical isolates to adhere to immobilized extracellular matrixes (ECMs) coating the walls of microtiter plates was examined by microscopy. The ECMs consisted of fibronectin, laminin, collagen types I, II, IV, and V, fibrinogen, and lactoferrin. With the exception of fibrinogen, each isolate showed a different level of adherence to each of the ECMs. No significant level of adherence to fibrinogen was observed for any isolate. The tissue-specific adhesive strains AS11, AS12, AS14, AS15, HT11, and HT12, which showed highly efficient adherence to human bladder carcinoma T24 cells and human bladder epithelial cells, showed strong adherence to fibronectin, laminin, and collagen type I, II, IV, and V ECMs, and the levels were greater than 104 cells/mm2 of well surface coated by ECM. None of the isolates that showed little adherence to human bladder carcinoma T24 cells showed efficient adherence to all the ECMs. The levels of adherence of gelatinase-producing isolates to the collagens were lower than the levels of adherence of gelatinase-negative isolates. When tissue-specific adhesive strains that adhered strongly to each ECM were preincubated with fibronectin, the adherence of the strains to fibronectin was inhibited, but the adherence of the strains to collagen type IV was not inhibited. Likewise, preincubation with collagen type IV inhibited adherence to collagen type IV but not adherence to fibronectin. All of the E. faecalis isolates were shown to carry the ace gene by PCR analysis performed with specific primers for collagen binding domain A of ace. The ace gene encodes Ace (adhesin of collagen from enterococci). The prtF gene of group A streptococci, which encodes the fibronectin binding protein of group A streptococci, was not detected in the tissue-specific adhesive strains by Southern analysis performed with the prtF probe of the Streptococcus pyogenes JRS4 strain. Mutants with altered collagen binding were isolated by insertion of Tn916 into the chromosome of tissue-specific adhesive strain AS14. The number of mutant adhesive bacterial cells that adhered to collagen and also to laminin was 1 or 2 orders lower than the number observed for the wild-type strain, but the level of adherence to fibronectin remained the same as that of the wild-type strain.
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LARSON, ADAM M., ANTHONY LEE, PO-FENG LEE, KAYLA J. BAYLESS, and ALVIN T. YEH. "ULTRASHORT PULSE MULTISPECTRAL NON-LINEAR OPTICAL MICROSCOPY." Journal of Innovative Optical Health Sciences 02, no. 01 (January 2009): 27–35. http://dx.doi.org/10.1142/s1793545809000292.
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Ultrashort pulse, multispectral non-linear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen. A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.
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Wilson, Richard, Susanna Freddi, Danny Chan, Kathryn S. E. Cheah, and John F. Bateman. "Misfolding of Collagen X Chains Harboring Schmid Metaphyseal Chondrodysplasia Mutations Results in Aberrant Disulfide Bond Formation, Intracellular Retention, and Activation of the Unfolded Protein Response." Journal of Biological Chemistry 280, no. 16 (February 4, 2005): 15544–52. http://dx.doi.org/10.1074/jbc.m410758200.
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Collagen X is a short chain collagen expressed specifically by the hypertrophic chondrocytes of the cartilage growth plate during endochondral bone formation. Accordingly,COL10A1mutations disrupt growth plate function and cause Schmid metaphyseal chondrodysplasia (SMCD). SMCD mutations are almost exclusively located in the NC1 domain, which is crucial for both trimer formation and extracellular assembly. Several mutations are expected to reduce the level of functional collagen X due to NC1 domain misfolding or exclusion from stable trimer formation. However, other mutations may be tolerated within the structure of the assembled NC1 trimer, allowing mutant chains to exert a dominant-negative impact within the extracellular matrix. To address this, we engineered SMCD mutations that are predicted either to prohibit subunit folding and assembly (NC1del10 and Y598D, respectively) or to allow trimerization (N617K and G618V) and transfected these constructs into 293-EBNA and SaOS-2 cells. Although expected to form stable trimers, G618V and N617K chains (like Y598D and NC1del10 chains) were secreted very poorly compared with wild-type collagen X. Interestingly, all mutations resulted in formation of an unusual SDS-stable dimer, which dissociated upon reduction. As the NC1 domain sulfhydryl group is not solvent-exposed in the correctly folded NC1 monomer, disulfide bond formation would result only from a dramatic conformational change. In cells expressing mutant collagen X, we detected significantly increased amounts of the spliced form of X-box DNA-binding protein mRNA and up-regulation of BiP, two key markers for the unfolded protein response. Our data provide the first clear evidence for misfolding of SMCD collagen X mutants, and we propose that solvent exposure of the NC1 thiol may trigger the recognition and degradation of mutant collagen X chains.
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Levy, A. D., J. Yang, and J. M. Kramer. "Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology." Molecular Biology of the Cell 4, no. 8 (August 1993): 803–17. http://dx.doi.org/10.1091/mbc.4.8.803.
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We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.
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Qiang, Shumin, Cheng Lu, and Fei Xu. "Disrupting Effects of Osteogenesis Imperfecta Mutations Could Be Predicted by Local Hydrogen Bonding Energy." Biomolecules 12, no. 8 (August 11, 2022): 1104. http://dx.doi.org/10.3390/biom12081104.
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Osteogenesis imperfecta(OI) is a disease caused by substitution in glycine residues with different amino acids in type I collagen (Gly-Xaa-Yaa)n. Collagen model peptides can capture the thermal stability loss of the helix after Gly mutations, most of which are homotrimers. However, a majority of natural collagen exists in heterotrimers. To investigate the effects of chain specific mutations in the natural state of collagen more accurately, here we introduce various lengths of side-chain amino acids into ABC-type heterotrimers. The disruptive effects of the mutations were characterized both experimentally and computationally. We found the stability decrease in the mutants was mainly caused by the disruption of backbone hydrogen bonds. Meanwhile, we found a threshold value of local hydrogen bonding energy that could predict triple helix folding or unfolding. Val caused the unfolding of triple helices, whereas Ser with a similar side-chain length did not. Structural details suggested that the side-chain hydroxyl group in Ser forms hydrogen bonds with the backbone, thereby compensating for the mutants’ decreased stability. Our study contributes to a better understanding of how OI mutations destabilize collagen triple helices and the molecular mechanisms underlying OI.
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Seedorf, H., I. N. Springer, E. Grundner-Culemann, H. K. Albers, A. Reis, H. Fuchs, M. Hrabe de Angelis, and Y. Açil. "Amelogenesis Imperfecta in a New Animal Model—a Mutation in Chromosome 5 (human 4q21)." Journal of Dental Research 83, no. 8 (August 2004): 608–12. http://dx.doi.org/10.1177/154405910408300805.
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Candidate genes for amelogenesis imperfecta (AI) and dentinogenesis imperfecta (DI) are located on 4q21 in humans. We tested our hypothesis that mutations in the portion of mouse chromosome 5 corresponding to human chromosome 4q21 would cause enamel and dentin abnormalities. Male C3H mice were injected with ethylnitrosourea (ENU). Within a dominant ENU mutagenesis screen, a mouse mutant was isolated with an abnormal tooth enamel (ATE) phenotype. The structure and ultrastructure of teeth were studied. The mutation was located on mouse chromosome 5 in an interval of 9 cM between markers D5Mit18 and D5Mit10. Homozygotic mutants showed total enamel aplasia with exposed dentinal tubules, while heterozygotic mutants showed a significant reduction in enamel width. Dentin of mutant mice showed a reduced content of mature collagen cross-links. We were able to demonstrate that a mutation on chromosome 5 corresponding to human chromosome 4q21 can cause amelogenesis imperfecta and changes in dentin composition.
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Bijian, Krikor, Tomoko Takano, Joan Papillon, Abdelkrim Khadir, and Andrey V. Cybulsky. "Extracellular matrix regulates glomerular epithelial cell survival and proliferation." American Journal of Physiology-Renal Physiology 286, no. 2 (February 2004): F255—F266. http://dx.doi.org/10.1152/ajprenal.00259.2003.
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Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-XL, compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased β1-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN.
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Lankhof, Hanneke, Conchi Damas, Marion E. Schiphorst, Martin J. W. IJsseldijk, Madelon Bracke, Jan J. Sixma, Tom Vink, and Philip G. de Groot. "Functional Studies on Platelet Adhesion With Recombinant von Willebrand Factor Type 2B Mutants R543Q and R543W Under Conditions of Flow." Blood 89, no. 8 (April 15, 1997): 2766–72. http://dx.doi.org/10.1182/blood.v89.8.2766.
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Abstract Type 2B von Willebrand disease (vWD) is characterized by the absence of the very high molecular weight von Willebrand factor (vWF ) multimers from plasma, which is caused by spontaneous binding to platelet receptor glycoprotein Ib (GPIb). We studied two mutations in the A1 domain at position 543 in which arginine (R) was replaced by glutamine (Q) or tryptophan (W), respectively. Both mutations were previously identified in vWD type 2B patients. The mutations R543Q and R543W were cloned into a eukaryotic expression vector and subsequently transfected in baby hamster kidney cells overexpressing furin (fur-BHK). Stable cell lines were established by which the mutants were secreted in the cell culture supernatant. The subunit composition and multimeric structure of R543Q and R543W were similar to wild-type (WT) vWF. The mutants showed a spontaneous binding to GPIb. R543Q and R543W showed normal binding to collagen type III or heparin. Both mutants supported platelet adhesion under conditions of flow, usually when preincubated on a collagen type III surface. A low dose (2.5% of the concentration present in normal pooled plasma) of recombinant R543Q or R543W added to normal whole blood inhibited platelet adhesion to collagen type III. No inhibition was found when vWF was used as an adhesive surface. These results indicate that point mutations identified in vWD type 2B cause bleeding symptoms by two mechanisms: (1) the mutants cause platelet aggregation, which in vivo is followed by removal of the aggregates leading to the loss of high molecular weight multimers and thrombocytopenia, (2) on binding to circulating platelets the mutants block platelet adhesion. Relatively few molecules are required for the latter effect.
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Gui, Tong, Hui-Feng Lin, Da-Yun Jin, Maureane Hoffman, David L. Straight, Harold R. Roberts, and Darrel W. Stafford. "Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo." Blood 100, no. 1 (July 1, 2002): 153–58. http://dx.doi.org/10.1182/blood.v100.1.153.
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Abstract Residue K5 in factor IX γ-carboxyglutamic acid (Gla) domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX–deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79% of the injected factor IX in the liver after 2 minutes, whereas 17% remained in circulation. In mice injected with K5A, 59% of the injected factor IX was found in liver and 31% was found in plasma. When we blocked the liver circulation before factor IX injection, 74% of K5A and 64% of K5R remained in the blood. When we treated the mouse with EDTA after injecting exogenous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of release from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical studies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from surgical specimens. Thus, we have demonstrated that factor IX binds in vivo to endothelial cell–collagen IV surfaces. Our results suggest that factor IX Gla-domain mediated binding to endothelial cells/collagen IV plays a role in controlling factor IX concentration in the blood.
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Matsumura, Hirokazu, Kiyoshi Kano, Caralina Marín de Evsikova, James A. Young, Patsy M. Nishina, Jürgen K. Naggert, and Kunihiko Naito. "Transcriptome analysis reveals an unexpected role of a collagen tyrosine kinase receptor gene, Ddr2, as a regulator of ovarian function." Physiological Genomics 39, no. 2 (October 2009): 120–29. http://dx.doi.org/10.1152/physiolgenomics.00073.2009.
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Mice homozygous for the smallie ( slie) mutation lack a collagen receptor, discoidin domain receptor 2 (DDR2), and are dwarfed and infertile due to peripheral dysregulation of the endocrine system of unknown etiology. We used a systems biology approach to identify biological networks affected by Ddr2slie/slie mutation in ovaries using microarray analysis and validate findings using molecular, cellular, and functional biological assays. Transcriptome analysis indicated several altered gene categories in Ddr2slie/slie mutants, including gonadal development, ovulation, antiapoptosis, and steroid hormones. Subsequent biological experiments confirmed the transcriptome analysis predictions. For instance, a significant increase of TUNEL-positive follicles was found in Ddr2slie/slie mutants vs. wild type, which confirm the transcriptome prediction for decreased chromatin maintenance and antiapoptosis. Decreases in gene expression were confirmed by RT-PCR and/or qPCR; luteinizing hormone receptor and prostaglandin type E and F receptors in Ddr2slie/slie mutants, compared with wild type, confirm hormonal signaling pathways involved in ovulation. Furthermore, deficiencies in immunohistochemistry for DDR2 and luteinizing hormone receptor in the somatic cells, but not the oocytes, of Ddr2slie/slie mutant ovaries suggest against an intrinsic defect in germ cells. Indeed, Ddr2slie/slie mutants ovulated significantly fewer oocytes; their oocytes were competent to complete meiosis and fertilization in vitro. Taken together, our convergent data signify DDR2 as a novel critical player in ovarian function, which acts upon classical endocrine pathways in somatic, rather than germline, cells.
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Nowak, Agata A., Kevin Canis, Anne Riddell, Michael A. Laffan, and Thomas A. J. McKinnon. "O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions." Blood 120, no. 1 (July 5, 2012): 214–22. http://dx.doi.org/10.1182/blood-2012-02-410050.
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AbstractWe have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D′ to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.
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Smethurst, Peter A., Lotta Joutsi-Korhonen, Marie N. O'Connor, Erica Wilson, Nicola S. Jennings, Stephen F. Garner, Yanjun Zhang, et al. "Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody." Blood 103, no. 3 (February 1, 2004): 903–11. http://dx.doi.org/10.1182/blood-2003-01-0308.
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Abstract Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)–like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.
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Arora, P. D., M. W. C. Chan, R. A. Anderson, P. A. Janmey, and C. A. McCulloch. "Separate Functions of Gelsolin Mediate Sequential Steps of Collagen Phagocytosis." Molecular Biology of the Cell 16, no. 11 (November 2005): 5175–90. http://dx.doi.org/10.1091/mbc.e05-07-0648.
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Collagen phagocytosis is a critical mediator of extracellular matrix remodeling. Whereas the binding step of collagen phagocytosis is facilitated by Ca2+-dependent, gelsolin-mediated severing of actin filaments, the regulation of the collagen internalization step is not defined. We determined here whether phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] regulation of gelsolin is required for collagen internalization. In gelsolin null fibroblasts transfected with gelsolin severing mutants, actin severing and collagen binding were strongly impaired but internalization and actin monomer addition at collagen bead sites were much less affected. PI(4,5)P2 accumulated around collagen during internalization and was associated with gelsolin. Cell-permeable peptides mimicking the PI(4,5)P2 binding site of gelsolin blocked actin monomer addition, the association of gelsolin with actin at phagosomes, and collagen internalization but did not affect collagen binding. Collagen beads induced recruitment of type 1 γ phosphatidylinositol phosphate kinase (PIPK1γ661) to internalization sites. Dominant negative constructs and RNA interference demonstrated a requirement for catalytically active PIPK1γ661 for collagen internalization. We conclude that separate functions of gelsolin mediate sequential stages of collagen phagocytosis: Ca2+-dependent actin severing facilitates collagen binding, whereas PI(4,5)P2-dependent regulation of gelsolin promotes the actin assembly required for internalization of collagen fibrils.
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Bijian, Krikor, Tomoko Takano, Joan Papillon, Ludmilla Le Berre, Jean-Louis Michaud, Chris R. J. Kennedy, and Andrey V. Cybulsky. "Actin cytoskeleton regulates extracellular matrix-dependent survival signals in glomerular epithelial cells." American Journal of Physiology-Renal Physiology 289, no. 6 (December 2005): F1313—F1323. http://dx.doi.org/10.1152/ajprenal.00106.2005.
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Adhesion of rat glomerular epithelial cells (GEC) to collagen activates focal adhesion kinase (FAK) and the Ras-extracellular signal-regulated kinase (ERK) pathway and supports survival (prevents apoptosis). The present study addresses the relationship between actin organization and the survival phenotype. Parental GEC (adherent to collagen) and GEC stably transfected with constitutively active mutants of mitogen-activated protein kinase kinase (R4F-MEK) or FAK (CD2-FAK) (on plastic) showed ERK activation, low levels of apoptosis, and a cortical distribution of F-actin. Parental GEC adherent to plastic showed increased apoptosis, disorganization of cortical F-actin, and formation of prominent stress fibers. Assembly of cortical F-actin was, at least in part, mediated via ERK. However, disruption of the actin cytoskeleton with cytochalasin D or latrunculin B in parental GEC (on collagen) and in GEC that express R4F-MEK or CD2-FAK (on plastic) decreased ERK activation and increased apoptosis. Expression of a constitutively active RhoA (L63RhoA) induced assembly of cortical F-actin, promoted ERK activation, and supplanted the requirement of collagen for survival. Adhesion of GEC to collagen increased phosphatidylinositol-4,5-bisphosphate (PIP2). Downregulation or sequestration of PIP2 by transfection with an inositol 5′-phosphatase or the plextrin-homology domain of phospholipase C-δ1 decreased F-actin content and survival. Moreover, overexpression of wild-type or K256E mutant α-actinin-4 with increased affinity for F-actin increased apoptosis. These results demonstrate a reciprocal relationship between collagen-induced cortical F-actin assembly and collagen-dependent survival signaling, including ERK activation. Appropriate remodeling of the actin cytoskeleton may be necessary for facilitating survival, as both disassembly and excessive crosslinking affect survival adversely.
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Cai, Liquan, Binh L. Phong, Alfred L. Fisher, and Zhou Wang. "Regulation of Fertility, Survival, and Cuticle Collagen Function by the Caenorhabditis elegans eaf-1 and ell-1 Genes." Journal of Biological Chemistry 286, no. 41 (August 31, 2011): 35915–21. http://dx.doi.org/10.1074/jbc.m111.270454.
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EAF2, an androgen-regulated protein, interacts with members of the ELL (eleven-nineteen lysine-rich leukemia) transcription factor family and also acts as a tumor suppressor. Although these proteins control transcriptional elongation and perhaps modulate the effects of other transcription factors, the mechanisms of their actions remain largely unknown. To gain new insights into the biology of the EAF2 and ELL family proteins, we used Caenorhabditis elegans as a model to explore the in vivo roles of their worm orthologs. Through the use of transgenic worms, RNAi, and an eaf-1 mutant, we found that both genes are expressed in multiple cell types throughout the worm life cycle and that they play important roles in fertility, survival, and body size regulation. ELL-1 and EAF-1 likely contribute to these activities in part through modulating cuticle synthesis, given that we observed a disrupted cuticle structure in ell-1 RNAi-treated or eaf-1 mutant worms. Consistent with disruption of cuticle structure, loss of either ELL-1 or EAF-1 suppressed the rol phenotype of specific collagen mutants, possibly through the control of dpy-3, dpy-13, and sqt-3 collagen gene expression. Furthermore, we also noted the regulation of collagen expression by ELL overexpression in PC3 human prostate cancer cells. Together, these results reveal important roles for the eaf-1 and ell-1 genes in the regulation of extracellular matrix components.
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Imanishi, Ayaka, Yuma Aoki, Masaki Kakehi, Shunsuke Mori, Tomomi Takano, Yukihiko Kubota, Hon-Song Kim, Yukimasa Shibata, and Kiyoji Nishiwaki. "Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans." PLOS ONE 15, no. 12 (December 2, 2020): e0240571. http://dx.doi.org/10.1371/journal.pone.0240571.
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During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.
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Bekkers, A. C. A. P. A., H. van der Vuurst, G. van Willigen, J. W. N. Akkerman, and H. M. Verheij. "Targeting of Porcine Pancreatic Phospholipase A2 to Human Platelets: Introduction of an RGD Sequence by Genetic Engineering." Thrombosis and Haemostasis 74, no. 04 (1995): 1138–44. http://dx.doi.org/10.1055/s-0038-1649894.
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SummaryThe possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A2 (PLA2) was tested by constructing a set of PLA2-mutants containing an Arg-Gly-Asp (RGD) sequence. One mutant was made with RGD as part of a surface-exposed loop (RGDloop). Four mutants were made with RGD as part of a C-terminal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x = 22,42 and 82 hydrophylic non-charged amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA2 and showed little binding to resting platelets. The binding of the native enzyme and RGDloop was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed stimulation-dependent binding to the platelet receptor GPIIb/IIIa, since GRGDS-peptide and a monoclonal antibody against the complex interfered with binding. In α-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of [3H]-arachidonic acid-labeled phospholipids. Stimulation with a combination of a-thrombin and collagen (known to expose phosphatidylserine) increased hydrolysis to 11%. Despite the membrane disruption, the cells did not leak lactate dehydrogenase. We conclude that PLA2 can be targeted to activated platelets by introducing RGD in a C-terminal extension with a minimum distance (42 amino acids) between RGD and the enzyme. However, more hydrolytic activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.
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Sylvestre, Patricia, Evelyne Couture-Tosi, and Michèle Mock. "Contribution of ExsFA and ExsFB Proteins to the Localization of BclA on the Spore Surface and to the Stability of the Bacillus anthracis Exosporium." Journal of Bacteriology 187, no. 15 (August 1, 2005): 5122–28. http://dx.doi.org/10.1128/jb.187.15.5122-5128.2005.
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ABSTRACT Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the ΔexsFA and ΔexsFA ΔexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The ΔexsFA and ΔexsFA ΔexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.
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Tsang, Kit Man, Russell H. Knutsen, Charles J. Billington, Eric Lindberg, Heiko Steenbock, Yi-Ping Fu, Amanda Wardlaw-Pickett, et al. "Copper-Binding Domain Variation in a Novel Murine Lysyl Oxidase Model Produces Structurally Inferior Aortic Elastic Fibers Whose Failure Is Modified by Age, Sex, and Blood Pressure." International Journal of Molecular Sciences 23, no. 12 (June 17, 2022): 6749. http://dx.doi.org/10.3390/ijms23126749.
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Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFβ-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease.
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Iwai, Leo K., Leo S. Payne, Maciej T. Luczynski, Francis Chang, Huifang Xu, Ryan W. Clinton, Angela Paul, et al. "Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants." Biochemical Journal 454, no. 3 (August 29, 2013): 501–13. http://dx.doi.org/10.1042/bj20121750.
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The present study characterizes integrin and DDR2 signalling networks activated by collagen. Using clustering approaches, DDR2-specific signalling components such as SHP-2 were identified. We further demonstrate that SHP-2 is phosphorylated by a subset of DDR2 lung cancer mutants.