Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells.

Syndecan-1 is a cell surface proteoglycan containing a highly conserved transmembrane and cytoplasmic domain, and an extracellular domain bearing heparan sulfate glycosaminoglycans. Through these domains, syndecan-1 is proposed to have roles in growth factor action, extracellular matrix adhesion, and cytoskeletal organization that controls cell morphology. To study the role of syndecan-1 in cell adhesion and cytoskeleton reorganization, mouse syndecan-1 cDNA was transfected into human Raji cells, a lymphoblastoid cell line that grows as suspended cells and exhibits little or no endogenous cell surface heparan sulfate. High expressing transfectants (Raji-Sl cells) bind to and spread on immobilized thrombospondin or fibronectin, which are ligands for the heparan sulfate chains of the proteoglycan. This binding and spreading as not dependent on the cytoplasmic domain of the core protein, is mutants expressing core proteins with cytoplasmic deletions maintain the ability to spread. The spreading is mediated through engagement of the syndecan-1 core protein, as the Raji-S 1 cells also bind to and spread on immobilized mAb 281.2, an antibody specific for the ectodomain of the syndecan-1 core protein. Spreading on the antibody is independent of the heparan sulfate glycosaminoglycan chains and can be inhibited by competition with soluble mAb 281.2. The spreading can be inhibited by treatment with cytochalasin D or colchicine. These data suggest that the core protein of syndecan-1 mediates spreading through the formation of a multimolecular signaling complex at the cell surface that signals cytoskeleton reorganization. This complex may form via intramembrane or extracellular interactions with the syndecan core protein.

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An investigation of genetic polymorphisms in Heparan Sulfate Proteoglycan core proteins and key modification enzymes in an Australian Caucasian multiple sclerosis population

10.21203/rs.2.23090/v1 ◽

2020 ◽

Author(s):

Rachel K Okolicsanyi ◽

Julia Bluhm ◽

Cassandra Miller ◽

Lyn R Griffiths ◽

Larisa M Haupt

Keyword(s):

Multiple Sclerosis ◽

Cell Surface ◽

Heparan Sulfate ◽

Demyelinating Disease ◽

Core Protein ◽

Association Studies ◽

International Studies ◽

Genome Wide Association Studies ◽

Nucleotide Polymorphisms ◽

Core Proteins

Abstract Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI-linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. Exostosin-1 (EXT1) and sulfatase-1 (SULF1) are two enzymes responsible for the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in initiation and polymerisation of the growing HS chain. SULF1 removes 6- O -sulfate groups from HS chains, thereby affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.

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Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2509 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2509-2518 ◽

Cited By ~ 92

Author(s):

A Rapraeger

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Heparan Sulfate ◽

Chondroitin Sulfate ◽

Transforming Growth Factor ◽

Core Protein ◽

Protein Characterization ◽

Tgf Beta ◽

The Core ◽

Cell Surface Proteoglycan

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.

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Alveolar type II cells synthesize hydrophobic cell-associated proteoglycans with multiple core proteins

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.263.3.l348 ◽

1992 ◽

Vol 263 (3) ◽

pp. L348-L356 ◽

Cited By ~ 2

Author(s):

W. M. Maniscalco ◽

M. H. Campbell

Keyword(s):

Extracellular Matrix ◽

Cell Surface ◽

Heparan Sulfate ◽

Core Protein ◽

Primary Cultures ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells ◽

Core Proteins

Type II alveolar epithelial cells interact with the extracellular matrix via cell surface receptors for matrix ligands. Cell surface proteoglycans, which are hydrophobic due to their membrane insertion domains, are one of several classes of molecules that may be receptors for matrix ligands. To analyze the hydrophobic proteoglycans synthesized by adult alveolar type II cells, we labeled these cells with 35SO4 and [3H]leucine in short-term primary cultures. Cell-associated hydrophobic proteoglycans and culture medium-derived proteoglycans were purified and characterized. Both the hydrophobic proteoglycans and medium-derived proteoglycans, which were not hydrophobic, had mainly heparan sulfate glycosaminoglycans. Analysis of core proteins of the hydrophobic proteoglycans showed three proteins, 47, 65, and 90 kDa. The 47- and 65-kDa core proteins were substituted only with heparan sulfate chains. The 90-kDa core protein was seen only after digestion with both heparitinase and chondroitin ABC lyase, suggesting it was a hybrid having both heparan sulfate and chondroitin-dermatan sulfate chains. These findings were confirmed by iodination of the core proteins. The hydrophobic cell-associated proteoglycans inserted into artificial liposomes, whereas the medium-derived molecules did not. These data document heterogeneity in core protein and glycosaminoglycan chains among hydrophobic proteoglycans synthesized in vitro by adult alveolar type II cells. These molecules may have diverse functions in regulating type II cell interaction with the extracellular matrix.

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Shedding of Syndecan-1 and -4 Ectodomains Is Regulated by Multiple Signaling Pathways and Mediated by a Timp-3–Sensitive Metalloproteinase

The Journal of Cell Biology ◽

10.1083/jcb.148.4.811 ◽

2000 ◽

Vol 148 (4) ◽

pp. 811-824 ◽

Cited By ~ 278

Author(s):

Marilyn L. Fitzgerald ◽

Zihua Wang ◽

Pyong Woo Park ◽

Gillian Murphy ◽

Merton Bernfield

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Signaling Pathways ◽

Proteolytic Activity ◽

Intracellular Signaling ◽

Protein Tyrosine Kinase ◽

Core Protein ◽

Ectodomain Shedding ◽

Core Proteins

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein–coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713–14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691–697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563–11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

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Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/35.10.2957423 ◽

1987 ◽

Vol 35 (10) ◽

pp. 1079-1088 ◽

Cited By ~ 152

Author(s):

K Hayashi ◽

M Hayashi ◽

M Jalkanen ◽

J H Firestone ◽

R L Trelstad ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Heparan Sulfate ◽

Plasma Cells ◽

Core Protein ◽

Basal Cells ◽

Alveolar Cells ◽

Electron Microscopic ◽

The Core ◽

Mouse Tissues

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.

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An investigation of genetic polymorphisms in Heparan Sulfate Proteoglycan core proteins and key modification enzymes in an Australian Caucasian multiple sclerosis population

10.21203/rs.2.23090/v2 ◽

2020 ◽

Author(s):

Rachel K Okolicsanyi ◽

Julia Bluhm ◽

Cassandra Miller ◽

Lyn R Griffiths ◽

Larisa M Haupt

Keyword(s):

Multiple Sclerosis ◽

Cell Surface ◽

Heparan Sulfate ◽

Demyelinating Disease ◽

Core Protein ◽

Association Studies ◽

International Studies ◽

Genome Wide Association Studies ◽

Nucleotide Polymorphisms ◽

Core Proteins

Abstract Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI-linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. The exostosin-1 (EXT1) and sulfatase-1 (SULF1) are key enzymes contributing to the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in initiation and polymerisation of the growing HS chain. SULF1 removes 6- O -sulfate groups from HS chains, affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.

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