Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.     

Plasmin- and thrombin-accelerated shedding of syndecan-4 ectodomain generates cleavage sites at Lys(114)-Arg(115) and Lys(129)-Val(130) bonds

Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of (35)S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys(114) downward arrowArg(115) and Lys(129) downward arrowVal(130) in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys(114) downward arrowArg(115) was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (Arg downward arrowGly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.     

Syndecan-1 ectodomain shedding is regulated by the small GTPase Rab5

The ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms.     

Syndecan-1 promotes Staphylococcus aureus corneal infection by counteracting neutrophil-mediated host defense

Many microbial pathogens subvert cell surface heparan sulfate proteoglycans (HSPGs) to infect host cells in vitro. The significance of HSPG-pathogen interactions in vivo, however, remains to be determined. In this study, we examined the role of syndecan-1, a major cell surface HSPG of epithelial cells, in Staphylococcus aureus corneal infection. We found that syndecan-1 null (Sdc1(-/-)) mice significantly resist S. aureus corneal infection compared with wild type (WT) mice that express abundant syndecan-1 in their corneal epithelium. However, syndecan-1 did not bind to S. aureus, and syndecan-1 was not required for the colonization of cultured corneal epithelial cells by S. aureus, suggesting that syndecan-1 does not mediate S. aureus attachment to corneal tissues in vivo. Instead, S. aureus induced the shedding of syndecan-1 ectodomains from the surface of corneal epithelial cells. Topical administration of purified syndecan-1 ectodomains or heparan sulfate (HS) significantly increased, whereas inhibition of syndecan-1 shedding significantly decreased the bacterial burden in corneal tissues. Furthermore, depletion of neutrophils in the resistant Sdc1(-/-) mice increased the corneal bacterial burden to that of the susceptible WT mice, suggesting that syndecan-1 moderates neutrophils to promote infection. We found that syndecan-1 does not affect the infiltration of neutrophils into the infected cornea but that purified syndecan-1 ectodomain and HS significantly inhibit neutrophil-mediated killing of S. aureus. These data suggest a previously unknown bacterial subversion mechanism where S. aureus exploits the capacity of syndecan-1 ectodomains to inhibit neutrophil-mediated bacterial killing mechanisms in an HS-dependent manner to promote its pathogenesis in the cornea.     

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin

Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.     

Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line.

The cloned rat fat pad endothelial cell (RFP-EC) line synthesizes anticoagulantly active heparan sulfate proteoglycans (HSPGact) and anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact), both of which exhibit 25-, 30-, and 50-kDa core proteins of extremely similar structure. The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kDa component from the HSPGinact core proteins demonstrate that the 30-kDa component is a previously unidentified species, designated as ryudocan, with the 25-kDa component representing a proteolytic degradation product, while the 50-kDa component is the rat homolog of syndecan (Saunders, S. Jalkanen, M., O'Farrell, S., and Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556). Specific oligonucleotide probes were obtained for ryudocan and syndecan by polymerase chain reaction, and the corresponding cDNAs were isolated from a RFP-EC library. The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions. In particular, ryudocan exhibits only three potential glycosaminoglycan attachment sites within the extracellular region while syndecan has five glycosaminoglycan attachment sites within the same domain. Both species are expressed in RFP-EC lines, primary rat aortic smooth muscle cells and primary rat skin fibroblast cells. The levels of ryudocan and syndecan mRNA were measured by quantitative polymerase chain reaction in primary microvascular endothelial cells and closely associated non-endothelial cells isolated by cell sorting. Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA.     

Identification of an invasion regulatory domain within the core protein of syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.     

Syndecans in wound healing, inflammation and vascular biology

Syndecans are heparan sulphate proteoglycans consisting of a type I transmembrane core protein modified by heparan sulphate and sometimes chondroitin sulphate chains. They are major proteoglycans of many organs including the vasculature, along with glypicans and matrix proteoglycans. Heparan sulphate chains have potential to interact with a wide array of ligands, including many growth factors, cytokines, chemokines and extracellular matrix molecules relevant to growth regulation in vascular repair, hypoxia, angiogenesis and immune cell function. This is consistent with the phenotypes of syndecan knock-out mice, which while viable and fertile, show deficits in tissue repair. Furthermore, there are potentially important changes in syndecan distribution and function described in a variety of human vascular diseases. The purpose of this review is to describe syndecan structure and function, consider the role of syndecan core proteins in transmembrane signalling and also their roles as co-receptors with other major classes of cell surface molecules. Current debates include potential redundancy between syndecan family members, the significance of multiple heparan sulphate interactions, regulation of the cytoskeleton and cell behaviour and the switch between promoter and inhibitor of important cell functions, resulting from protease-mediated shedding of syndecan ectodomains.     

Syndecan-1-mediated cell spreading requires signaling by alphavbeta3 integrins in human breast carcinoma cells

Syndecans are cell surface heparan sulfate proteoglycans with regulatory roles in cell adhesion, proliferation, and differentiation [Annu. Rev. Biochem. 68 (1999) 729]. While the syndecan heparan sulfate chains are essential for matrix binding, less is known about the signaling role of their core proteins. To mimic syndecan-specific adhesion, MDA-MB-231 mammary carcinoma cells were plated on antibodies against syndecan-4 or syndecan-1. While cells adherent via syndecan-4 spread, cells adherent via syndecan-1 do not. However, cells adherent via syndecan-1 can be induced to spread by Mn(2+), suggesting that activation of a beta(1) or beta(3) integrin partner is required. Surprisingly, pretreatment of cells with a function-activating beta(1) antibody does not induce spreading, whereas function-blocking beta(1) integrin antibodies do, suggesting involvement of a beta(1)-to-beta(3) integrin cross-talk. Indeed, blockade of beta(1) integrin activation induces alpha(v)beta(3) integrin activation detectable by soluble fibrinogen binding. Spreading in response to syndecan-1 is independent of integrin-ligand binding. Furthermore, competition with soluble murine syndecan-1 ectodomain, which does not disrupt cell adhesion, nonetheless blocks the spreading mechanism. These data suggest that the ectodomain of the syndecan-1 core protein directly participates in the formation of a signaling complex that signals in cooperation with alpha(v)beta(3) integrins; signaling via this complex is negatively regulated by beta(1) integrins.     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

Shedding of syndecan-1 by stromal fibroblasts stimulates human breast cancer cell proliferation via FGF2 activation

The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.     

Constitutive and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site

Syndecan-1 is a developmentally regulated cell surface heparan sulfate proteoglycan (HSPG). It functions as a coreceptor for a variety of soluble and insoluble ligands and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and recently feeding behavior. The extracellular domain of syndecan-1 is proteolytically cleaved at a juxtamembrane site by tissue inhibitor of metalloprotease-3 (TIMP-3)-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts syndecan-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. We found that replacing syndecan-1 juxtamembrane amino acid residues A243-S-Q-S-L247 with human CD4 amino acid residues can completely block PMA-induced syndecan-1 ectodomain shedding. Furthermore, using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), we identified the proteolytic cleavage site of syndecan-1 as amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine NMuMG cells. Finally, we show that basal cleavage of syndecan-1 utilizes the same in vivo site as the in vitro site. Indeed, as predicted, transgenic mice expressing the syndecan-1/CD4 cDNA do not shed the syndecan-1 ectodomain in vivo. These results suggest that the same cleavage site is utilized for basal syndecan-1 ectodomain shedding both in vitro from NMuMG and CHO cells and in vivo.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.     

Endogenous attenuation of allergic lung inflammation by syndecan-1

The airway plays a vital role in allergic lung diseases by responding to inhaled allergens and initiating allergic inflammation. Various proinflammatory functions of the airway epithelium have been identified, but, equally important, anti-inflammatory mechanisms must also exist. We show in this study that syndecan-1, the major heparan sulfate proteoglycan of epithelial cells, attenuates allergic lung inflammation. Our results show that syndecan-1-null mice instilled with allergens exhibit exaggerated airway hyperresponsiveness, glycoprotein hypersecretion, eosinophilia, and lung IL-4 responses. However, administration of purified syndecan-1 ectodomains, but not ectodomain core proteins devoid of heparan sulfate, significantly inhibits these inflammatory responses. Furthermore, syndecan-1 ectodomains are shed into the airway when wild-type mice are intranasally instilled with several biochemically distinct inducers of allergic lung inflammation. Our results also show that syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17) implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress allergen-induced accumulation of Th2 cells in the lung through their heparan sulfate chains. Together, these findings uncover an endogenous anti-inflammatory mechanism of the airway epithelium where syndecan-1 ectodomains attenuate allergic lung inflammation via suppression of CC chemokine-mediated Th2 cell recruitment to the lung.     

Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins

Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.     

2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection

Ablation of syndecan-1 in mice is a gain of function mutation that enables mice to significantly resist infection by several bacterial pathogens. Syndecan-1 shedding is induced by bacterial virulence factors, and inhibition of shedding attenuates bacterial virulence, whereas administration of purified syndecan-1 ectodomain enhances virulence, suggesting that bacteria subvert syndecan-1 ectodomains released by shedding for their pathogenesis. However, the pro-pathogenic functions of syndecan-1 ectodomain have yet to be clearly defined. Here, we examined how syndecan-1 ectodomain enhances Staphylococcus aureus virulence in injured mouse corneas. We found that syndecan-1 ectodomain promotes S. aureus corneal infection in an HS-dependent manner. Surprisingly, we found that this pro-pathogenic activity is dependent on 2-O-sulfated domains in HS, indicating that the effects of syndecan-1 ectodomain are structure-based. Our results also showed that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate motifs inhibit S. aureus killing by antimicrobial factors secreted by degranulated neutrophils, but does not affect intracellular phagocytic killing by neutrophils. Immunodepletion of antimicrobial factors with staphylocidal activities demonstrated that CRAMP, a cationic antimicrobial peptide, is primarily responsible for S. aureus killing among other factors secreted by degranulated neutrophils. Furthermore, we found that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate units potently and specifically inhibit S. aureus killing by synthetic CRAMP. These results provide compelling evidence that a specific subclass of sulfate groups, and not the overall charge of HS, permits syndecan-1 ectodomains to promote S. aureus corneal infection by inhibiting a key arm of neutrophil host defense.     

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.