Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion

Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan- sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.     

Masking of anionic sites by deposits in lamina rara externa in immune complex nephritis in rats.

Alterations in glomerular basement membrane (GBM) anionic sites associated with immune deposits (ID) were observed using polyethyleneimine (PEI) as a cationic probe in serum sickness nephritis induced by egg albumin (EA). The anionic sites were fewer in number than in other GBM segments and were irregular in distribution in most, but not all, of the segments of the GBM with ID on the epithelial side of the lamina densa (LD). The disappearance of anionic sites was obvious in areas where the internal aspects of the lamina rara externa (LRE) of the GBM were occupied by ID, even if the ID were very small. In contrast, the disappearance of anionic sites was not evident, even though no change in anionic sites was found in some areas, where the ID had departed from the internal aspects of the LRE and a pale band was seen between the ID and the LD. Further, PEI aggregates, showing localization of anionic sites, were seen within the low density ID, but no PEI aggregates were seen within the high density ID. The results suggest that: 1) whether or not ID induce the disappearance of anionic sites is independent of the size of the ID, but is dependent on the density of and the place occupied by the ID, and 2) the ID seem to induce the disappearance of anionic sites by masking rather than destroying them.     

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.     

Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate proteoglycan in human skin and other basement membranes

A variety of heparan sulfate proteoglycans (HSPG) have been identified on cell surfaces and in basement membrane (BM). To more fully characterize HSPG in human skin BM, we used two monoclonal antibodies (MAb) directed against epitopes of the core protein of a high molecular weight HSPG isolated from murine EHS tumor. Indirect immunofluorescence revealed linear distribution of HSPG within all skin BM, and within BM of all other human organs investigated. In a study of the ontogeny of HSPG in human skin BM, HSPG was detectable as early as 54 gestational days, comparable with other ubiquitous BM components, such as laminin and type IV collagen. Immunoelectron microscopy on adult skin and neonatal foreskin showed staining primarily within the lamina densa (LD) and sub-lamina densa regions of the dermoepidermal junction (DEJ) and vascular BM. In neonatal foreskin, additional staining was noted of basilar cytoplasmic membranes of keratinocytes, endothelial cells, and pericytes. We conclude that the core protein of a high molecular weight HSPG is ubiquitous in human BM, appears in fetal skin on or before 54 days, and is present primarily in the regions of the LD and sub-LD.     

Localization of proteoheparan sulfate in rat aorta

This study describes the distribution of heparan sulfate proteoglycan ( HSPG ) within the rat aorta using immunocytochemical (biotin-avidin-peroxidase) and immuno-electron microscopy (125I-autoradiography). Heparan sulfate proteoglycan was isolated from a basement membrane producing mouse EHS sarcoma ( Hassell et al. 1980) and used to generate antisera in rabbits. Light microscopic observations revealed intense immunostaining of the intima and media of normal aorta, adventitial vasa vasorum, and aortic intimal fibromuscular thickenings induced by experimental injury (balloon de-endothelialization). Immunoelectron microscopy using 125I labeled antibodies to HSPG revealed that proteoheparan sulfate was localized to the amorphous layer of basement membrane below aortic and capillary endothelium. In addition, labeled anti- HSPG could be localized to the external lamina surrounding the smooth muscle cells in the hyperplastic intima. These studies reveal that antibodies prepared against a proteoheparan sulfate isolated from a basement membrane producing EHS sarcoma cross react with basement membrane structures within the aortic wall. Furthermore, these results demonstrate that the basement membranes beneath aortic and capillary endothelium and the external lamina surrounding aortic smooth muscle cells contain a heparan sulfate proteoglycan that is antigenically similar.     

Preservation of fixed anionic sites in the GBM in the acute proteinuric phase of cationic antigen mediated in-situ immune complex glomerulonephritis in the rat

Cationic antigens have been observed to bind with the negatively charged glomerular basement membrane (GBM). Using the cationic reagent polyethyleneimine (PEI), the distribution of glomerular anionic sites was evaluated ultrastructurally in the early stage (2 h-day 7) of cationic antigen mediated in-situ immune complex formation type glomerulonephritis (GN) in the rat. Renal perfusion via the renal artery with 100 micrograms of cationized human IgG(pI greater than 9.5), followed by the i.v. injection of specific antibodies, led to an initial increase in urinary albumin excretion, subsequent massive globulinuria and the formation of numerous subepithelial deposits on day 7. The most striking alteration in glomerular anionic sites was observed on the epithelial cell surface coat; the PEI deposition on the epithelial cell surface was almost identical to that in control glomeruli at 2 and 4 h after the induction of GN; thereafter, on day 7, a broad loss of anionic sites was observed on flattened epithelial foot processes. In contrast, fixed anionic sites of the laminae rarae of the GBM showed no apparent alterations in the distribution and number from 2 h to day 7 and did not disappear even in the lamina rara externa adjacent to subepithelial deposits. These findings not only show that fixed anionic sites of the GBM, in contrast to the rapid decrease in those of the epithelial cell surface, are not completely neutralized or destroyed even in GN, in which cationic antigen participates in the in-situ formation of GBM-deposits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

Summary The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.     

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.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

Changes in the glomerular filtration barrier during aging in rats

In this work, we analysed histochemical, biochemical and functional modifications of the glomerular basement membrane (GBM), occurring for aging, in the Rat. The results suggest an increase of collagenous components and a decrease of sulfated glucosaminoglycans as a function of age. In other respects, fixed anionic sites of the GBM, disclosed by colloidal iron, are almost exclusively restricted to the laminae rarae in one month-old rats, whereas the marker appears randomly scattered among the lamina densa in 12 month-old animals. These changes could be the cause of increased permeability of the GBM during aging.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

Reduced content and abnormal distribution of anionic sites (acid proteoglycans) in the diabetic glomerular basement membrane

Sulfated proteoglycans (fixed anionic sites) on the glomerular basement membrane (GBM) of kidneys from diabetic and nondiabetic patients have been demonstrated by electron microscopy using polycationic dyes (ruthenium red, polyethyleneimine). These substances were used for immersion fixation of renal biopsy specimens. The thickened GBM of diabetics revealed a reduced proteoglycan content within both the narrowed laminae rarae, where normally particles were seen at 60 nm intervals. Proteinuria was observed in all such cases, but no immunopathological alterations of the basement membranes were seen. With both tracer substances anionic sites were also demonstrated in different segments of the thickened lamina densa in diabetics. In polyethyleneimine-treated biopsies some segments of the membrane showed increased anionic moieties at the junction of the basement membrane and the epithelial and endothelial cell membranes. These are probably acid glycoproteins linked to the cell membrane and the synthesis of these basement membrane components may represent a compensatory mechanism seeking to restore normal permeability.     

A comparison of anionic sites in the glomerular basement membranes from different classes of fishes

Summary Cationized ferritin was injected into the circulatory system of teleosts, the sea raven and Atlantic eelpout, and into elasmobranchs, the spiny dogfish and the skate, to determine if the glomerular basement membranes (GBM) from these different groups of fishes possess anionic binding sites similar to those present in the GBM of mammals. The distribution of cationized ferritin was the same in all fishes listed. Cationized ferritin was localized only in the GBM and the mesangial matrix. The regular distribution of cationized ferritin within the laminae rarae (60 nm intervals) was taken as evidence of the presence of anionic binding sites. Cationized ferritin did not bind to the glomerular capillary endothelium, nor was any of it localized at the base of the slit diaphragms of the foot processes of the podocytes. The distribution of binding sites in the GBM of these fishes is similar to that in another teleost, the winter flounder, and in a cyclostome, the hagfish.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basement membrane proteoglycans and anionic sites in the fetal rat kidney during late gestation

Proteoglycans were studied in developing rat fetal kidney using cytological and biochemical techniques. These compounds were detected with immunoperoxidase in the nephron basement membranes from the earliest stages of differentiation. In the glomerular basement membrane, immunostaining appeared as both diffuse and granular deposits, as long as this membrane consisted of loose material; however, as soon as a three-layered membrane had formed, staining was confined to the laminae rarae as regularly arranged granules. The same pattern of staining was observed during differentiation of the basement membrane of the proximal tubule. In Bowman's capsule, immunostaining appeared as granules, which were sparsely distributed in the developing glomerulus and then regularly lined the stacked laminae when differentiation was complete. In all basement membranes, anionic sites (disclosed by polyethyleneimine) were colocated with immunostained granular deposits. Total glycosaminoglycan content gradually increased from the beginning of metanephros development to birth. During this period, the relative proportions of glycosaminoglycans changed: heparan sulfate increased and hyaluronic acid decreased as differentiation proceeded. The possible relationship between morphological observations and biochemical changes in glycosaminoglycan content is discussed.     

Localization of proteoheparan sulfate in rat aorta

Summary This study describes the distribution of heparan sulfate proteoglycan (HSPG) within the rat aorta using immunocytochemical (biotin-avidin-peroxidase) and immunoelectron microscopy (¹²⁵I-autoradiography). Heparan sulfate proteoglycan was isolated from a basement membrane producing mouse EHS sarcoma (Hassell et al. 1980) and used to generate antisera in rabbits. Light microscopic observations revealed intense immunostaining of the intima and media of normal aorta, adventitial vasa vasorum, and aortic intimal fibromuscular thickenings induced by experimental injury (balloon de-endothelialization). Immunoelectron microscopy using ¹²⁵I labeled antibodies to HSPG revealed that proteoheparan sulfate was localized to the amorphous layer of basement membrane below aortic and capillary endothelium. In addition, labeled anti-HSPG could be localized to the external lamina surrounding the smooth muscle cells in the hyperplastic intima. These studies reveal that antibodies prepared against a proteoheparan sulfate isolated from a basement membrane producting EHS sarcoma cross react with basement membrane structures within the aortic wall. Furthermore, these results demonstrate that the basement membranes beneath aortic and capillary endothelium and the external lamina surrounding aortic smooth muscle cells contain a heparan sulfate proteoglycan that is antigenically similar.     

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)