Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts

Heparitinase digestion of the hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) of fetal human lung fibroblasts yields core proteins of various sizes: i.e. monomeric core proteins of 125, 90, 64, 48, and 35 kDa and a disulfide-linked dimeric core protein composed of approximately 35-kDa subunits. By immunizing BALB/c mice with liposome-incorporated HSPG, we have obtained a total of five anti-HSPG monoclonal antibodies (Mabs, i.e. Mabs S1, 1C7, 2E9, 6G12, and 10H4) with different specificities. Polyacrylamide gel electrophoresis of 125I-labeled membrane HSPG immunoprecipitated with these Mabs revealed that Mabs 1C7 and 2E9 bind only membrane HSPG which yield a 125-kDa core protein after heparitinase digestion, whereas Mab S1-bound HSPG yield a 64-kDa core protein, and Mabs 6G12 and 10H4 retain membrane HSPG with a 48-kDa core protein. Western blotting of the heparitinase-digested proteoglycans and immunostaining with the Mabs confirmed this pattern of reactivity. However, in this assay, Mabs 6G12 and 10H4 also detected a minor approximately 90-kDa core protein in addition to the 48-kDa core protein. Except perhaps for the 10H4 epitope, the epitopes recognized by these Mabs appear to be part of the peptide moieties as they resisted complete deglycosylation of the HSPG with trifluoromethanesulfonic acid. Since these data were inconsistent with a direct relationship between the major core proteins, the 48-, 64-, and 125-kDa core proteins were immunopurified and further compared by peptide mapping with Staphylococcus aureus protease V8, trypsin, and CNBr cleavage. Clearly distinct peptide patterns were obtained for the three different core proteins. These results imply that the 48-, 64-, and the 125-kDa membrane HSPG core proteins of human lung fibroblasts are derived from distinct proteoglycans.     

Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms

Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.     

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.     

Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes.

1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably.     

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.     

Identification of cell surface molecules that interact with pseudorabies virus.

The alphaherpesvirus pseudorabies virus (PrV) has been shown to attach to cells by interaction between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). A secondary binding step requires gD and presumably another, hitherto unidentified cellular receptor. By use of a virus overlay protein binding assay (VOPBA), cosedimentation analyses, and affinity chromatography, we identified three species of cell membrane constituents that bind PrV. By treatment with EDTA, peripheral HSPGs of very high apparent molecular mass (>200 kDa) could be extracted from Madin-Darby bovine kidney cells. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzymatic digestion with heparinase or papain. Cosedimentation analyses indicated that binding between PrV and high-molecular-weight HSPG depended on the presence of gC in the virion. In addition, adsorption of radiolabeled PrV virions to cells could be inhibited by the addition of purified high-molecular-weight HSPG. By using urea extraction buffer, a second species of HSPG of approximately 140 kDa could be solubilized. Binding of PrV to this HSPG in the VOPBA was also dependent on the presence of heparan sulfate, since reactivity was abolished after suppression of glycosaminoglycan biosynthesis with NaClO3 and after heparinase treatment. In addition to HSPG, in cellular membrane extracts obtained by treatment with mild detergent, a 85-kDa membrane protein was demonstrated to bind PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents that bind PrV: a peripheral HSPG of high molecular weight, an integral HSPG of approximately 140 kDa, and an integral membrane protein of 85 kDa. It is tempting to speculate that interaction between PrV and the two species of HSPG mediates primary attachment of PrV and that the 85-kDa protein is involved in a subsequent attachment step.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Syndecans reside in sphingomyelin-enriched low-density fractions of the plasma membrane isolated from a parathyroid cell line

Background

Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction.

Methodology/Principal Findings

Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [³⁵S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([³⁵S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [³⁵S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30–33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase.

Conclusions/Significance

Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.     

Characterization of heparan sulfate proteoglycan from calf lens capsule and proteoglycans synthesized by cultured lens epithelial cells. Comparison with other basement membrane proteoglycans

After extraction with 4 M guanidinium chloride and purification by DEAE-cellulose chromatography, the heparan sulfate proteoglycan (HSPG) of calf anterior lens capsule was found to consist of two immunologically related components (Mr = 340,000 and 250,000) which upon deglycosylation with trifluoromethanesulfonic acid yielded core proteins with Mr values of 170,000 and 145,000. The heparan sulfate chains were uniform in size (Mr = 14,000) and manifested a clustering of sulfate groups in a peripheral domain. From the decrease in Mr observed after heparitinase digestion, it could be estimated that 6 and 11 glycosaminoglycan chains were present in the Mr = 250,000 and 340,000 components respectively. The occurrence of N-linked oligosaccharides was evident from the size difference of the heparitinase- and trifluoromethane-sulfonic acid-treated proteoglycans (approximately 20 kDa), as well as from the presence of a substantial number of mannose residues; furthermore, interaction of the capsule proteoglycan with Bandeiraea simplicifolia I suggested that these carbohydrate units contains terminal alpha-D-Gal groups. Cultured lens epithelial cells deposited a single [35S]sulfate-labeled proteoglycan into their matrix (Mr = 400,000) which was immunologically related to the lens capsule proteoglycan and contained only heparan sulfate chains. In addition to this component, the medium from these cells contained an immunologically unrelated HSPG (Mr = 150,000) as well as a chondroitin sulfate proteoglycan (Mr = 240,000). Examination of bovine glomeruli indicated that, in addition to the previously described 200-kDa HSPG, an immunologically related 350-kDa component was also present. This size heterogeneity, which is comparable to that seen in the lens capsule, is most readily attributable to proteolytic processing of a precursor molecule. Studies with polyclonal antibodies demonstrated only limited cross-reactivities between the Engelbreth-Holms-Swarm proteoglycan and the components from lens capsule and glomerular basement membrane; since even the latter two differed somewhat in their antigenic sites, it would appear that cell- and species-dictated genetic differences as well as post-translational events contribute to the diversity observed in basement membrane HSPGs.     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. II. Structural characterization of the keratan sulfate and dermatan sulfate proteoglycans from corneal stroma

Radioisotopically labeled proteoglycans were isolated from a 4 M guanidine HCl, 2% Triton X-100 extract of corneal stroma from day 18 chicken embryos by anion-exchange chromatography. Two predominant proteoglycans in the sample were separated by octyl-Sepharose chromatography using a gradient elution of detergent in 4 M guanidine HCl. One proteoglycan had an overall mass of approximately 125 kDa, a single dermatan sulfate chain (approximately 85-90% chondroitin 4-sulfate, low iduronate content) of approximately 65 kDa, and a core protein after chondroitinase ABC digestion of approximately 45 kDa which also contained one to three N-linked oligosaccharides and one O-linked oligosaccharide. The other proteoglycan had an overall size of approximately 100 kDa, two to three keratan sulfate chains of approximately 15 kDa each, and a core protein following keratanase digestion of approximately 51 kDa which included two to three N-linked but no O-linked oligosaccharides. A larger size, a greater overall hydrophobicity (as measured by its interaction with octyl-Sepharose) and an absence of O-linked oligosaccharides argue that this core protein is a distinct gene product from the core protein of the dermatan sulfate proteoglycan.     

Nerve Growth Factor-Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells

Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC12 cells after 3 days of culture was in the range of 90-190 kDa for the intact form (Kav = 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90-190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120-150 kDa and 120-190 kDa (Kav = 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav = 0.22 on Sephacryl S-300 [40-84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.     

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.     

Membrane associated proteoglycans in rat testicular peritubular cells

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG) Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (K_av=0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.Abbreviations PG Proteoglycans - CSPG Chondroitin Sulfate Proteoglycans - HSPG Heparan Sulfate Proteoglycans - HSCSPG Heparan and Chondroitin Sulfate Proteoglycans - DNAse I Deoxyribonuclease I - DMEM Dulbeccos modified Eagle's medium - H/D HAM F12/DMEM - ECM Extracellular Matrix - PBS Phosphate Buffered Saline - PI Phosphatidylinositol - GPI Glycosyl Phosphatidylinositol - PI-PLC Phosphatidylinositol Specific Phospholipase C - TBS Tris Buffered Saline - STI Soybean Trypsin Inhibitor - GAG Glycosaminoglycans - HA Hyaluronic Acid     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.     

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.     

A Chondroitin/Dermatan Sulfate Form of CD44 Is a Receptor for Collagen XIV (Undulin)

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of¹²⁵I-labeled fibroblast cell surface proteins on collagen XIV–Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97–105 kDa after chondroitinase ABC digestion and of 60–70 kDa after further treatment withN-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.     

Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen

Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.     

Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4- labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI- PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC- released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.