Auswahl der wissenschaftlichen Literatur zum Thema „Human endosulfatases“

Abstract Human sulfatases Hsulf-1 and Hsulf-2 are extracellular endosulfatases that specifically remove 6-O sulfates from heparan sulfate chains. Published studies have shown that expression of Hsulf-1 in some tumor cell lines diminishes their response to heparin-binding growth factors (e.g., FGF-2, HGF) leading to speculation that these enzymes may regulate tumor growth. To examine this possibility in vivo, the cDNA for either Hsulf-1 or Hsulf-2 was expressed in the human myeloma cell line CAG. Disaccharide composition analysis of heparan sulfate chains isolated from the surface of the transfected cells reveals that 6-O sulfation of trisulfated disaccharides is diminished by 70% and 50% on cells expressing Hsulf-1 and Hsulf-2, respectively, as compared to control cells transfected with empty vector only. When stimulated with exogenous FGF-2, downstream signaling as measured by the level of phosphorylated ERK1/2, is reduced in HSulf-1 or Hsulf-2 transfectants as compared to controls. To determine if Hsulf-1 and Hsulf-2 regulate myeloma tumor cell growth in vivo, cells were injected subcutaneously into the left flank of SCID mice. Monitoring of whole animal tumor burden by analysis of human kappa light chain present in the serum revealed that tumors formed from cells expressing either Hsulf-1 or Hsulf-2 exhibited a dramatic inhibition of tumor growth as compared to tumors formed from control cells (p<0.0004). Upon sacrifice, mean weights of primary tumors were 0.46 g (n=12), 0.18 g (n=17) and 2.44 g (n=16) for Hsulf-1, Hsulf-2 and control tumors, respectively (p<0.0001). To confirm that the endosulfatase enzymes were active in vivo, assembly of the FGF-2 ternary signaling complex (FGF-2/heparan sulfate/FGF Receptor 1) was analyzed on frozen tissue sections of the tumors. Incorporation of the FGFR1 into the complex requires the presence of 6-O sulfate groups in the endogenous heparan sulfate chains. Experiments show that FGF-2 binds equally well to the heparan sulfate present in all tumors, but the FGFR1 is not incorporated into the complex on tumors expressing either Hsulf-1 or Hsulf-2. This indicates that the Sulfs are active in vivo and that this activity alters heparin-binding growth factor signaling of the tumor cells. Additionally, we find that the elevation of Sulf expression causes a marked increase in extracellular matrix (ECM) deposition within tumors which may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These studies indicate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of myeloma tumors in vivo.

Colorectal cancer is the third most common cancer worldwide, accounting for more than 610,000 mortalities every year. Prognosis of patients is highly dependent on the disease stage at diagnosis. Therefore, it is crucial to investigate molecules involved in colorectal cancer tumorigenesis, with possible use as tumor markers. Heparan sulfate proteoglycans are complex molecules present in the cell membrane and extracellular matrix, which play vital roles in cell adhesion, migration, proliferation, and signaling pathways. In colorectal cancer, the cell surface proteoglycan syndecan-2 is upregulated and increases cell migration. Moreover, expression of syndecan-1 and syndecan-4, generally antitumor molecules, is reduced. Levels of glypicans and perlecan are also altered in colorectal cancer; however, their role in tumor progression is not fully understood. In addition, studies have reported increased heparan sulfate remodeling enzymes, as the endosulfatases. Therefore, heparan sulfate proteoglycans are candidate molecules to clarify colorectal cancer tumorigenesis, as well as important targets to therapy and diagnosis.

Here we report the cloning of a full-length cDNA encoding the human ortholog (HSulf-1) of the developmentally regulated putative sulfatases QSulf-1 (Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D. M., and Emerson, C. P., Jr. (2001)Science293, 1663–1666) and RSulfFP1 (Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002)Genes Cells7, 173–185) as well as a cDNA encoding a closely related protein, designated HSulf-2. We have also obtained cDNAs for the mouse orthologs of both Sulfs. We demonstrate that the proteins encoded by both classes of cDNAs are endoproteolytically processed in the secretory pathway and are released into conditioned medium of transfected CHO cells. We demonstrate that the mammalian Sulfs exhibit arylsulfatase activity with a pH optimum in the neutral range; moreover, they can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Taken together, our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interactions of heparan sulfate proteoglycans in the extracellular microenvironment.

Abstract The heparan sulfate proteoglycan syndecan-1 is expressed by myeloma cells and shed into the myeloma microenvironment. High levels of shed syndecan-1 in myeloma patient sera correlate with poor prognosis and studies in animal models indicate that shed syndecan-1 is a potent stimulator of myeloma tumor growth and metastasis. Overexpression of extracellular endosulfatases, enzymes which remove 6-O sulfate groups from heparan sulfate chains, diminishes myeloma tumor growth in vivo. Together, these findings identify syndecan-1 as a potential target for myeloma therapy. Here, 3 different strategies were tested in animal models of myeloma with the following results: (1) treatment with bacterial heparinase III, an enzyme that degrades heparan sulfate chains, dramatically inhibited the growth of primary tumors in the human severe combined immunodeficient (SCID-hu) model of myeloma; (2) treatment with an inhibitor of human heparanase, an enzyme that synergizes with syndecan-1 in promoting myeloma progression, blocked the growth of myeloma in vivo; and (3) knockdown of syndecan-1 expression by RNAi diminished and delayed myeloma tumor development in vivo. These results confirm the importance of syndecan-1 in myeloma pathobiology and provide strong evidence that disruption of the normal function or amount of syndecan-1 or its heparan sulfate chains is a valid therapeutic approach for this cancer.

Pan-cancer analysis of TCGA and CPTAC (proteomics) data shows that SULF1 and SULF2 are oncogenic in a number of human malignancies and associated with poor survival outcomes. Our studies document a consistent upregulation of SULF1 and SULF2 in HNSC which is associated with poor survival outcomes. These heparan sulfate editing enzymes were considered largely functional redundant but single-cell RNAseq (scRNAseq) shows that SULF1 is secreted by cancer-associated fibroblasts in contrast to the SULF2 derived from tumor cells. Our RNAScope and patient-derived xenograft (PDX) analysis of the HNSC tissues fully confirm the stromal source of SULF1 and explain the uniform impact of this enzyme on the biology of multiple malignancies. In summary, SULF2 expression increases in multiple malignancies but less consistently than SULF1, which uniformly increases in the tumor tissues and negatively impacts survival in several types of cancer even though its expression in cancer cells is low. This paradigm is common to multiple malignancies and suggests a potential for diagnostic and therapeutic targeting of the heparan sulfatases in cancer diseases.

Les endosulfatases humaines HSulf-1 et HSulf-2 catalysent la 6-O-désulfatation de l'héparane sulfate (HS), un composant majeur de la matrice extracellulaire, essentiel à la communication cellulaire. La modification des motifs de sulfatation de l'HS par HSulfs module ses propriétés d'interaction. Bien qu'ayant des activités enzymatiques similaires in vitro, les HSulfs présentent in vivo une dualité fonctionnelle, notamment dans le cancer où HSulf-1 est généralement anti-oncogénique et HSulf-2 pro-oncogénique. Ce travail vise à caractériser les propriétés structurales et fonctionnelles des HSulfs pour comprendre le lien entre leurs caractéristiques structurales uniques et leur impact fonctionnel. En combinant approches analytiques (chromatographie, électrophorèse, spectrométrie de masse) et techniques d'imagerie (photométrie de masse, microscopie à force atomique), nous nous sommes focalisé sur trois aspects principaux : (1) les éléments structuraux influençant l'oligomérisation de HSulf-2, en particulier sa chaîne de GAG (CS/DS) et son domaine hydrophile (HD), (2) les caractéristiques fonctionnelles de l'activité endosulfatase des HSulfs, notamment leur spécificité de substrat, via le développement d'un nouveau test évaluant leur réactivité envers des séquences oligosaccharidiques spécifiques et (3) l'évaluation d'oligosaccharides fonctionnalisés par des sulfamates comme inhibiteurs. Les résultats obtenus apportent apportent de nouvelles perspectives sur les relations structure-fonction des HSulfs et leur ciblage thérapeutique
Human endosulfatases HSulf-1 and HSulf-2 catalyze the 6-O-desulfation of the polysaccharide heparan sulfate (HS), a key cellular component used by cells as an external communication tool. By editing the sulfation patterns of HS, HSulfs regulate its biomolecular interaction properties. Despite their similar enzymatic activities in vitro, HSulfs often display form-dependent discrepancies in vivo, particularly in cancer, where HSulf-1 generally demonstrates anti-oncogenic properties while HSulf-2 shows pro-oncogenic effects. This work aims to characterize the structural and functional properties of HSulfs to understand the link between their unique structural characteristics and functional involvement. Using complementary analytical approaches, including chromatography, electrophoresis, mass spectrometry, and single molecule Imaging techniques, we investigated three main aspects: (1) the structural elements influencing HSulf-2 oligomerization, with particular emphasis on its CS/DS GAG chain and hydrophilic domain (HD), (2) the functional characteristics of HSulf endosulfatase activity, particularly substrate specificity, using a novel assay to assess HSulfs' reactivity towards specific oligosaccharide sequences and (3) the evaluation of sulfamate-modified oligosaccharides as potential specific inhibitors of HSulf activity. The results obtained provide new insights into our understanding of HSulf structure-function relationships and contribute to the growing knowledge base for potential therapeutic approaches targeting these enzymes

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy Medicinal Chemistry and Natural Products." Discipline: Medicinal Chemistry and Natural Products; Department/School: Pharmacy.

Les Héparanes Sulfates (HS) sont de polysaccharides complexes impliqués dans de nombreux processus biologiques. La structure des HS est contrôlée à la surface cellulaire par une famille particulière d‟endosulfatases extracellulaires, les Sulfs. Les Sulfs modifient dramatiquement les propriétés fonctionnelles des HS et sont impliqués dans de nombreux processus physiopathologiques, notamment le cancer. Ces enzymes se composent de deux domaines: un domaine catalytique (CAT) contenant le site actif et un domaine basique hydrophile (HD) responsable de la liaison aux HS. Le but de mon projet de thèse est de caractériser les propriétés structurales et fonctionnelles de la forme humaine HSulf-2, qui demeure à ce jour très mal connues. Dans ce cadre, nous avons tout d‟abord étudié les mécanismes de reconnaissance enzyme/substrat et caractérisé deux nouveaux motifs de reconnaissance des HS sur ces enzymes, responsable de leur activité. En utilisant des oligosaccharides naturels et synthétiques, nous avons aussi démontré que le domaine HD n'est pas essentiel pour la reconnaissance des HS, mais est permet une désulfatation processive et orientée du polysaccharide. De plus, nous avons identifié un tétrasaccharide comme étant la taille oligosaccharidique minimale requise pour l'activité de HSulf-2. Nos résultats nous ont permis de proposer un nouveau modèle décrivant le processus de désulfatation du HS par HSulf-2. D'autre part, nous avons montré que HSulf-2 est un protéoglycane, car il contient une modification post-traductionnelle unique (chaîne CS de Chondoitin Sulfate) sur son domaine HD. Cette chaîne diminue l'activité enzymatique et la liaison aux HS in vitro. Dans le microenvironnement tumoral, en utilisant un modèle de tumeur mammaire orthotopique murin, nous avons montré que la chaîne CS est libérée par protéolyse, conduisant à l'activation de HSulf-2, augmentant la capacité des tumeurs à se développer et à se transformer en métastase. Finalement, nous avons réalisé une étude structurale des Sulfs. Nous avons choisi d‟étudier séparément les deux domaines (CAT et HD). Des essais de cristallogenèse ont été menés pour le domaine CAT afin de résoudre sa structure par cristallographie aux rayons X, mais n‟ont pu aboutir. En ce qui concerne le HD, nous avons mis en place un protocole de production et de purification de HD d‟une manière recombinante et nous avons initiés une étude par RMN ainsi que d'autres techniques biophysiques afin de caractériser structuralement le domaine et d'identifier les sites de liaison aux HS. Nos résultats préliminaires suggèrent que la HD est un domaine non structuré, à l'exception de ses parties N- et C-terminales. L‟ensemble de ces travaux devrait nous permettre de mieux comprendre ces importants mécanismes de régulation des HS et de d‟envisager de nouvelles stratégies anticancéreuses ciblant les Sulfs
Heparan Sulfate (HS) are complex polysaccharides involved in many biological processes. The structure of HS is regulated at the cell surface by unique extracellular endosulfatases, the Sulfs. Sulfs dramatically change HS functional properties, thereby being implicated in many physiopathological processes including cancer. Sulfs features two domains: a catalytic domain (CAT) that comprises the active site, and an hydrophilic basic domain (HD) responsible for HS binding. The aim of my PhD project is to characterize the structural and the functional properties of the human for HSulf-2, which remains poorly understood. In this context, we have first studied the enzyme/substrate recognition mechanisms. We identified two novel HS binding motifs on these enzymes implicated in their activity. In addition, using natural and synthetic oligosaccharides, we demonstrated that the HD is not essential for HS recognition, but is directs the processive and orientated desulfation of the polysaccharide. Moreover, we showed that a tetrasaccharide is the minimal oligosaccharide size required for HSulf-2 activity. Our results enabled us to propose a new model depicting the desulfation process of HS by the Sulfs. Second, we have shown that HSulf-2 is a proteoglycan, given that it harbors a unique PTM (Chondroitin Sulfate, CS chain) on its HD domain. This chain decreases enzyme activity and HS binding in vitro. In the tumoral microenvironment, using a murine orthotropic mammary tumor model, we showed that the CS chain is lost by proteolytic processing, leading to the activation of HSulf-2, and the promotion of tumor growth, vascularization and metastasis. Finally, we have undertaken the structural characterization of the Sulfs. For this, we decided to study separately the two domains found in these enzymes (CAT and HD). Crystallogenesis assays were undertaken for the CAT domain to solve its structure by X-ray crystallography, but were unsuccessful. Regarding the HD, we set up a protocol of production and purification of recombinant HD and we initiated NMR studies and other biophysics analyses in order to structurally characterize the domain and to identify the HS binding sites. Our preliminary results suggest that the HD is an unstructured domain, except for its N- and C-terminal parts. Overall, our data provide significant insights into this critical regulatory step of HS function