Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.     

Analysis of glycosaminoglycans in bovine retinal microvessel basement membrane

Glycosaminoglycans (GAG) were isolated from bovine retinal microvessel basement membrane (RMV-BM) and quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to nanogram amounts of heparan and chondroitin sulfates. Treatment of osmotically lysed retinal microvessels with the ionic detergent deoxycholate (DOC), required for liberation of the extracellular matrix for plasma membrane lipoproteins and purification of the insoluble matrix, solubilized less than 5% of the GAG in the water-insoluble material. Total GAG content in the DOC-insoluble basement membranes was approx. 0.52 micrograms/mg dry weight; about 70% of the measurable GAG was resistant to both chondroitinase ABC and chondroitinase AC digestion and was sensitive to nitrous acid treatment, indicating its heparan sulfate nature. Cellulose acetate electrophoresis revealed two bands, one of which had an electrophoretic mobility similar to heparan sulfate standard and was sensitive to nitrous acid; the other migrated in the same position as chondroitin sulfate standard and was sensitive to chondroitinase ABC and chondroitinase AC digestion. These results provide evidence that RMV-BM contains chondroitin sulfate(s) as well as heparan sulfate, and offer the first quantitative analysis of GAG in this extracellular matrix.     

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth—Holm—Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

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.     

Multiple heparan sulfate proteoglycans synthesized by a basement membrane producing murine embryonal carcinoma cell line

The murine embryonal carcinoma derived cell line M1536-B3 secretes the basement membrane components laminin and entactin and, when grown in bacteriological dishes, produces and adheres to sacs of basement membrane components. Heparan sulfate proteoglycans have been isolated from these sacs, the cells, and the medium. At least three different heparan sulfate proteoglycans are produced by these cells as determined by proteoglycan size, glycosaminoglycan chain length, and charge density. The positions of the N- and O-sulfate groups in the glycosaminoglycan chains from each proteoglycan appear to be essentially the same despite differences in the size and culture compartment locations of the heparan sulfate proteoglycan. Additionally, small quantities of chondroitin sulfate proteoglycans are found in each fraction and copurify with each heparan sulfate proteoglycan. Because this cell line appears to synthesize at least three different heparan sulfate proteoglycans which are targeted to different final locations (basement membrane, cell surface, and medium), this will be a useful system in which to study the factors which determine final heparan sulfate proteoglycan structures and culture compartment targeting and the possible effects of the protein core(s) on heparan sulfate carbohydrate chain synthesis and secretion.     

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth-Holm-Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.     

Heparan sulfate proteoglycan from human and equine glomeruli and tubules

1. Proteoglycans were isolated from human and equine glomeruli or tubules by guanidine extraction and anion exchange chromatography. 2. These proteoglycan preparations contained about equal amounts of heparan sulfate and chondroitin sulfates. 3. During the preparation of glomerular or tubular basement membranes the main part of proteoglycans (greater than 50%) was extracted in the salt extract. Chondroitin sulfate proteoglycan was mainly found in the water and salt extracts of glomeruli and tubules, heparan sulfate proteoglycan in the deoxycholate extracts and the basement membranes. 4. The glomerular basement membrane (GBM) contains about 12% (human) or 20% (equine) of the proteoglycans of the total glomerulus. They consist of greater than 70% (equine) or 80% (human) of heparan sulfate. 5. Heparan sulfate proteoglycan was isolated from the proteoglycan preparations of human or equine glomeruli and tubules by additional treatment with nucleases and chondroitinase ABC followed by CsCl gradient centrifugation. 6. Protein accounts for about 40% (dry weight) of the heparan sulfate proteoglycans. Their amino acid composition is characterized by a high content of glycine, but 3-hydroxyproline, 4-hydroxyproline and hydroxylysine are lacking. 7. The biochemical characteristics of the heparan sulfate proteoglycan of human or equine glomeruli or tubules differ from that isolated from rat glomeruli by their higher protein content and their amino acid composition. The significance of these differences is discussed.     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.     

Characterization of a rat yolk sac carcinoma cell line

A continuous cell line was established from an experimentally induced rat yolk sac carcinoma. In the early passages both visceral and parietal yolk sac carcinoma were present (designated L1). When the cell line was reestablished in culture after serial transplantations in rats, only parietal yolk sac carcinoma could be identified (designated L2). This cell line expresses parietal yolk sac endoderm characteristics in that it synthesizes basement membrane components, in particular, laminin, but also entactin, collagen IV, and heparan sulfate proteoglycan. In addition, a noncartilage chondrotin sulfate proteoglycan is synthesized. This rat yolk sac carcinoma cell line L2 will be a valuable model for the study of basement membrane components.     

Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.     

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on celulose acetate membrane (Separax), in succession.Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases (Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2–5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.     

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.     

Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.     

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red

Summary Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digetion tests withStreptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase.Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10–20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10–80 nm in diameter and fine filaments 3–4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.