Gotowa bibliografia na temat „CMP-sialic acid transport proteins”

Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.

Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

ABSTRACTEngagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA−, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA−, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA− in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

HeLa cells arrested in prometaphase were pulse-labeled with [35S]methionine and chased in the absence of nocodazole to allow passage through mitosis and into G1. Transport of histocompatibility antigen (HLA) molecules to the medial- and trans-Golgi cisternae was measured by monitoring the resistance to endoglycosidase H and the acquisition of sialic acid residues, respectively. Transport to the plasma membrane was measured using neuraminidase to remove sialic acid residues on surface HLA molecules. The half-time for transport to each of these compartments was about 65-min longer in cells progressing out of mitosis than in G1 cells. This delay was only 5-min longer than the half-time for the fall in histone H1 kinase activity suggesting that inactivation of the mitotic kinase triggers the resumption of protein transport. The half-time for reassembly of the Golgi stack, measured using stereological procedures, was also 65 min, suggesting that both transport and reassembly are triggered at the same time. However, since reassembly was complete within 5 min, whereas HLA took 25 min to reach the medial-cisterna, we can conclude that the Golgi stack has reassembled by the time HLA reaches it.

ABSTRACT How receptors control virus infection is poorly understood. Polyomavirus (Py) binds to the sialic acid-galactose moiety on receptors to gain entry into host cells and cause infection. We previously demonstrated that the sialic acid-galactose-containing glycolipids called gangliosides GD1a and GT1b promote Py infection, in part, by sorting the virus from the endolysosomes to the endoplasmic reticulum (ER), a critical infection route. Whether these glycolipids act as Py entry receptors, however, is not clear. Additionally, as the majority of glycoproteins also harbor terminal sialic acid-galactose residues, their roles in Py infection are also not well established. Using a ganglioside-deficient cell line, we show that GD1a is the functional entry receptor for Py. GD1a binds to Py on the plasma membrane, and the receptor-virus complex is internalized and transported to the late endosomes and then the ER to initiate infection. In contrast, our findings indicate that glycoproteins act as decoy receptors, restricting the ER transport and infection of Py. Thus, glycolipids and glycoproteins, two major constituents of the plasma membrane, execute opposing functions in regulating infection by a defined virus.

ABSTRACT Platelet binding by Streptococcus gordonii strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by S. gordonii Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of S. gordonii to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of S. gordonii Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in secA2. Hsa export can be enhanced by complementation with secA2 from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.

ABSTRACT We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.

The solute/sodium symporter family (SSS family; TC 2.A.21; SLC5) consists of integral membrane proteins that use an existing sodium gradient to drive the uphill transport of various solutes, such as sugars, amino acids, vitamins, or ions across the membrane. This large family has representatives in all three kingdoms of life. The human sodium/iodide symporter (NIS) and the sodium/glucose transporter (SGLT1) are involved in diseases such as iodide transport defect or glucose-galactose malabsorption. Moreover, the bacterial sodium/proline symporter PutP and the sodium/sialic acid symporter SiaT play important roles in bacteria–host interactions. This review focuses on the physiological significance and structural and functional features of prokaryotic members of the SSS family. Special emphasis will be given to the roles and properties of proteins containing an SSS family domain fused to domains typically found in bacterial sensor kinases.

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.

Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of α(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.

Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of ?(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.
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Doctor of Philosophy (PhD)
Institute for Glycomics
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Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria.

Most glyco-engineering approaches used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We investigated whether the over expression of nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT), would be a means to improve the sialylation process in CHO cells. We hypothesized that increasing the expression of the CMP-SAT in the cells would increase the transport of the CMP-sialic acid in the Golgi lumen, hence increasing the intra-lumenal CMP-sialic acid pool, and resulting in a possible increase in sialylation extent of proteins being produced. We report the construction of a CMP-SAT expression vector which was used for transfection into CHO-IFNγ, a CHO cell line producing human IFNγ. This resulted in approximately 2 to 5 times increase in total CMP-SAT expression in some of the positive clones as compared to untransfected CHO-IFNγ, as determined using real-time PCR analysis. This in turn concurred with a 9.6% to 16.3% percent increase in site sialylation. This engineering approach has thus been identified as a novel means of improving sialylation in recombinant glycoprotein therapeutics. This strategy can be utilized feasibly on its own, or in combination with existing sialylation improvement strategies. It is believed that such multi-prong approaches are required to effectively manipulate the complex sialylation process, so as to bring us closer to the goal of producing recombinant glycoproteins of high and consistent sialylation from mammalian cells.
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In multicellular organisms, thin lipid membranes serve as semipermeable barriers between aqueous compartments. The plasma membrane of the cell separates the cytoplasm from the extracellular space; endothelial cell membranes separate the blood within the vascular space from the rest of the tissue. Properties of the lipid membrane are critically important in regulating the movement of molecules between these aqueous spaces. While certain barrier properties of membranes can be attributed to the lipid components, accessory molecules within the cell membrane—particularly transport proteins and ion channels—control the rate of permeation of many solutes. Transport proteins permit the cell to regulate the composition of its intracellular environment in response to extracellular conditions. The relationship between membrane structure, membrane function, and cell physiology is an area of active, ongoing study. Our interest here is practical: what are the basic mechanisms of drug movement through membranes and how can one best predict the rate of permeation of an agent through a membrane barrier? To answer that question, this section presents rates of permeation measured in some common experimental systems and models of membrane permeation that can be used for prediction. The external surface of the plasma membrane carries a carbohydrate-rich coat called the glycocalyx; charged groups in the glycocalyx, which are provided principally by carbohydrates containing sialic acid, cause the surface to be negatively charged. On average, the plasma membrane of human cells contains, by mass, 50% protein, 45% lipid, and 5% carbohydrate. Given the mass ratio of protein to lipid is ~ 1 : 1, and assuming reasonable values for the average molecular weight and cross-sectional area for each type of molecule (50 × Mw,lipid = Mw,protein; Alipid = 50 Å2 and Aprotein = 1,000 Å2), the area fraction of protein on a typical membrane is ~ 33%. The lipid composition varies in membranes from different cells depending on the type of cell and its function. In addition, the outermost monolayer of lipids, called the outer leaflet, has a different lipid composition from the inner leaflet.

The external surface of the cell consists of a phospholipid bilayer which carries a carbohydrate-rich coat called the glycocalyx; ionizable groups within the glycocalyx, such as sialic acid (N-acetyl neuraminate), contribute a net negative charge to the cell surface. Many of the carbohydrates that form the glycocalyx are bound to membrane-associated proteins. Each of these components— phospholipid bilayer, carbohydrate-rich coat, membrane-associated protein—has distinct physicochemical characteristics and is abundant. Plasma membranes contain ∼50% protein, ∼45% lipid, and ∼5% carbohydrate by weight. Therefore, each component influences cell interactions with the external environment in important ways. Cells can become attached to surfaces. The surface of interest may be geometrically complex (for example, the surface of another cell, a virus, a fiber, or an irregular object), but this chapter will focus on adhesion between a cell and a planar surface. The consequences of cell–cell adhesion are considered further in Chapter 8 (Cell Aggregation and Tissue Equivalents) and Chapter 9 (Tissue Barriers to Molecular and Cellular Transport). The consequences of cell–substrate adhesion are considered further in Chapter 7 (Cell Migration) and Chapter 12 (Cell Interactions with Polymers). Since the growth and function of many tissue-derived cells required attachment and spreading on a solid substrate, the events surrounding cell adhesion are fundamentally important. In addition, the strength of cell adhesion is an important determinant of the rate of cell migration, the kinetics of cell–cell aggregation, and the magnitude of tissue barriers to cell and molecule transport. Cell adhesion is therefore a major consideration in the development of methods and materials for cell delivery, tissue engineering, and tissue regeneration. The most stable and versatile mechanism for cell adhesion involves the specific association of cell surface glycoproteins, called receptors, and complementary molecules in the extracellular space, called ligands. Ligands may exist freely in the extracellular space, they may be associated with the extracellular matrix, or they may be attached to the surface of another cell. Cell–cell adhesion can occur by homophilic binding of identical receptors on different cells, by heterophilic binding of a receptor to a ligand expressed on the surface of a different cell, or by association of two receptors with an intermediate linker. Cell–matrix adhesion usually occurs by heterophilic binding of a receptor to a ligand attached to an insoluble element of the extracellular matrix.