Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associated heparan sulfate proteoglycans of lung fibroblasts. Prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts

Heparitinase treatment of cell surface-associated heparan sulfate proteoglycans (HSPG) of human lung fibroblasts reveals core proteins with apparent Mr values of 125,000, 90,000, 64,000, 48,000 and 35,000 (Lories, V., De Boeck, H., David, G., Cassiman, J.-J., and Van den Berghe, H. (1987) J. Biol. Chem. 262, 854-859). The 90- and 48-kDa core proteins share the epitope of the monoclonal antibody 6G12 which was used to screen a human lung fibroblast expression cDNA library. Rescreening of the libraries yielded clone 48K5 with an insert of 3439 base pairs. Polyclonal antibodies were raised in rabbits against a fragment of the protein encoded by the 48K5 cDNA different from the part carrying the 6G12 epitope. These antibodies specifically recognize the 90- and 48-kDa core proteins on Western blots of total cellular extracts of human lung fibroblast HSPG. The specific reactivity of the polyclonal antiserum confirms the identity of the 48K5 clone and further distinguishes the 48- and the 90-kDa core proteins, which do share the 6G12-defined epitope and at least one additional antigenic determinant with the 48K5 cDNA-encoded protein, from the 125-, 64-, and 35-kDa core proteins of cell surface HSPG of human lung fibroblasts which do not react with either antibody preparation. The protein encoded by the 48K5 clone contains a stop-transfer sequence indicative of an integral membrane protein and three potential glycosaminoglycan attachment sites. The 48K5 clone detects two major poly(A)+ RNA species in human lung fibroblasts presumably generated by the use of alternative polyadenylation signals. The 48K5 gene was mapped to chromosome 8q23 by in situ hybridization and hybridization to DNA of somatic cell hybrids.     

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.     

Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody.

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).     

Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures

Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(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     

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.     

Two forms of plasma membrane-intercalated heparan sulfate proteoglycan in rat ovarian granulosa cells. Labeling of proteoglycans with a photoactivatable hydrophobic probe and effect of the membrane anchor-specific phospholipase C

The structures of cell-associated heparan sulfate (HS) proteoglycans and their interaction with the plasma membrane was studied using rat ovarian granulosa cell culture. HS proteoglycans were either metabolically labeled by incubating cell cultures with [3H] leucine and [35S]sulfate or labeled in plasma membrane preparations with a photoactivatable reagent, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), a compound which has been shown to selectively label the hydrophobic membrane-binding domains of several proteins. After purification of HS proteoglycans from the labeled cell cultures or from the labeled membrane preparations by repeated Q-Sepharose ion exchange chromatography in 8 M urea, they were analyzed by Superose 6 gel filtration and octyl-Sepharose chromatography both in 4 M guanidine HCl. The results indicated that the HS proteoglycans were labeled with 125I and therefore have an intramembranous domain. Phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol membrane anchors, released approximately 25% of the 35S-labeled HS proteoglycans from the cell surface as well as 20-30% of the 125I-label from the 125I-TID-labeled HS proteoglycans. These data indicate that a subpopulation of HS proteoglycans are intercalated into the plasma membrane through a linkage structure involving phosphatidylinositol. Phospholipase C-resistant, 125I-labeled HS proteoglycans represent those species inserted into membrane through an intercalated peptide sequence. Core protein size of phosphatidylinositol-anchored species estimated by polyacrylamide gel electrophoresis after heparitinase digestion was approximately 80 kDa, and it was significantly larger than that of the directly intercalated species (approximately 70 kDa).     

Unique glycosylation of three keratan sulfate proteoglycan isoforms

Recent work demonstrates isoforms of bovine corneal keratan sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of keratan sulfate with endo-beta-galactosidase. Keratan sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three keratan sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal keratan sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.     

Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis

(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.     

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 and characterization of three immunodominant structural proteins of fowlpox virus

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.     

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.     

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.     

Isolation and partial characterization of lumican and decorin from adult chicken corneas. A keratan sulfate-containing isoform of decorin is developmentally regulated.

The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.     

Immunocytochemical,electrophoresis, and immunoblot analysis of Heliothis virescens gap junctions isolated in the presence and absence of protease inhibitors

Gap junction-enriched fractions were prepared from larvae of the tobacco budworm Heliothis virescens using the NaOH procedure in the presence or absence of protease inhibitors and were analyzed by SDS-PAGE, immunoblotting and EM immunocytochemistry. Protease inhibitor fractions contained a 48-kDa protein in addition to the 10 proteins in fractions with and without inhibitors. Three polyclonal antibodies were used as probes for gap junction plaques and proteins: R16, against an 40-kDa candidate gap junction protein from Drosophila melanogaster; R17, against the 40-kDa candidate gap junction protein from H. virescens; and R18AP, an affinity purified antibody against a consensus sequence of N-terminal amino acids 2–21 of the H. virescens 40-kDa protein. R16, R17, and R18AP stain the 40- and 48-kDa proteins, R16 and R18AP stain a 64-kDa protein, and R16 stains an 30-kDa protein in the absence of inhibitors. Inclusion of protease inhibitors had no effect on gap junction ultrastructure. R16 and R17 label gap junction plaques in crude membrane and NaOH fractions, whereas R18AP exhibits only a low level of reactivity with gap junctions in crude membrane fractions and none with gap junctions in NaOH fractions. The results show that the 30-, 40-, 48- and 64-kDa proteins are immunologically related and are associated with gap junctions in H. virescens, the N-terminus of the 40-kDa protein is relatively inaccessible or easily lost, and the 48-kDa protein is protease-sensitive.     

Cell-surface heparan sulfate and heparan-sulfate/chondroitin-sulfate hybrid proteoglycans of mouse mammary epithelial cells

The hydrophobic cell-surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion-exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan-sulfate chains, larger proteoglycans yielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I-labeled proteoglycans with heparitin-sulfate lyase and chondroitin ABC lyase yielded core proteins of approximately 93 kDa, approximately 85 kDa and approximately 38 kDa. Comparison with single enzyme digestions identified the 93-kDa and 85-kDa cores as components of hybrid proteoglycans that carried both heparan-sulfate and chondroitin-sulfate chains. Immunoblotting indicated that the 93-kDa and 85-kDa cores shared the epitope defined by monoclonal antibody 281-2. The 38-kDa core, in contrast, carried only heparan-sulfate chains and lacked the 281-2 epitope. Preparations enriched in heparan sulfate or in heparan-sulfate/chondroitin-sulfate hybrid proteoglycans were obtained by N-desulfation and ion-exchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin-sulfate and nearly half of the heparan-sulfate residues of the proteoglycans showed a similar polydispersity of heparan-sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan-sulfate chains. These hybrids contained heparan-sulfate and chondroitin-sulfate chains in similar molar amounts. Analysis of 125I-labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85-kDa core protein that carries a single chondroitin-sulfate chain and a single heparan-sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related, 93-kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38-kDa core and carried only heparan-sulfate chains. The significance of the co-existence of hybrid and heparan-sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.     

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.     

Characterization of outer membrane proteins in Chlamydia trachomatis LGV serovar L2

We used a photoactivatable, lipophilic reagent, 3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of Chlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.