Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-beta

Transforming growth factors beta 1 and beta 2 bind with high affinity to the core protein of a 250-350-kD cell surface proteoglycan. This proteoglycan (formerly referred to as the type III TGF-beta receptor) coexists in many cells with the receptor implicated in TGF-beta signal transduction (type I TGF-beta receptor), but its function is not known. We report here that soluble TGF-beta-binding proteoglycans are released by several cell types into the culture media, and can be found in serum and extracellular matrices. As has been shown for the membrane-bound form, the soluble proteoglycans have a heterogeneous core protein of 100-120 kD that carries chondroitin sulfate and/or heparan sulfate glycosaminoglycan chains and a small amount of N-linked carbohydrate. The membrane-bound form of this proteoglycan is hydrophobic and associates with liposomes, whereas the soluble forms lack a membrane anchor and do not associate with liposomes. Differences in the electrophoretic migration of the soluble and membrane forms of this proteoglycan suggest additional structural differences in their core proteins and glycosaminoglycan chains. These soluble and membrane-bound proteoglycans, for which we propose the name "betaglycans," might play distinct roles in pericellular retention, delivery, or clearance of activated TGF-beta.     

Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes.

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.     

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.     

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.     

Age-related changes in the proteoglycans of human skin

Skin undergoes dramatic age-related changes in its mechanical properties, including changes in tissue hydration and resiliency. Proteoglycans are macromolecular conjugates of protein and carbohydrate (glycosaminoglycan) which are involved in these tissue properties. In order to examine whether age-related changes in skin proteoglycans may contribute to the age-related changes in the mechanical properties of skin, proteoglycans from human skin of various ages were extracted and analyzed. Samples were obtained from two different fetal ages, from mature skin, and from senescent skin. As a function of age, there is a decrease in the proportion of large chondroitin sulfate proteoglycans (versican) and a concomitant increase in the proportion of small dermatan sulfate proteoglycans (decorin). Based on reactivity with antibodies to various chondroitin sulfate epitopes, fetal versican differs from the versican found in older skin with respect to the chondroitin sulfate chains. Also, the decorin of fetal skin is slightly larger, while the decorin of older skin shows greater polydispersity in both its size and its charge to mass ratio. There are also age-related differences in the size and polydispersity of the core proteins of decorin. The most pronounced change in skin proteoglycans is the appearance in mature skin of a proteoglycan which is smaller than decorin, but which has the same amino terminal amino acid sequence as decorin. This small proteoglycan is abundant in mature skin and may be a catabolic fragment of decorin or an alternatively spliced form of decorin. In light of the known ability of decorin to influence collagen fibrillogenesis and fibril diameter, the appearance of this small decorin-related proteoglycan may have a significant effect on skin elasticity. The observation that proteoglycans in skin show dramatic age-related differences suggests that these changes may be involved in the age-related changes in the physical properties of skin.     

Nonlysosomal processing of cell-surface heparan sulfate proteoglycans. Studies of I-cells and NH4Cl-treated normal cells

The metabolism of cell-associated proteoglycans, labeled in the glycosaminoglycan portion with 35SO2-4, was studied in normal skin fibroblasts (SL66 cells), NH4Cl-treated SL66 fibroblasts, and in I-cells derived from patients with mucolipidosis II. Kinetic data from label-chase experiments and gel filtration analysis of the molecular weight distribution of the radiolabeled glycosaminoglycans indicated that I-cells and NH4Cl-treated normal fibroblasts (a) internalize cell surface proteoglycans, (b) remove glycosaminoglycan chains from proteoglycan core proteins, and (c) degrade heparan sulfate glycosaminoglycan chains via an endoglycosidic activity. These processes occur with rates comparable to those in normal fibroblasts. The data are consistent with the hypothesis that the glycosaminoglycan chains of cell-surface proteoglycans are separated from the protein cores in a nonlysosomal compartment prior to the transport of these chains to lysosomes for degradation. These observations also raise the possibility that this early step in separation of glycosaminoglycan chains from protein cores may serve to regulate the levels of glycosaminoglycan-free core protein observed in various cells.     

Isolation and characterization of proteoglycans synthesized by adult human gingival fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human gingivae were isolated and characterized. The largest medium proteoglycan was excluded from Sepharose CL-4B but not from Sepharose CL-2B; it was recovered in the most-dense density gradient fraction and identified as a chondroitin sulfate proteoglycan. The medium contained two smaller proteoglycans; one contained predominantly chondroitin sulfate proteoglycan, while the other was comprised predominantly of dermatan sulfate proteoglycan and was quantitatively the major species. The largest proteoglycan in the cell layer fraction, excluded from both Sepharose CL-2B and Sepharose CL-4B, was found in the least-dense density gradient fraction and contained heparan sulfate and chondroitin sulfate proteoglycan. It could be further dissociated by treatment with detergent, suggesting an intimate association with cell membranes. Two other proteoglycan populations of intermediate size were identified in the cell layer extracts which contained variable proportions of heparan sulfate, dermatan sulfate, or chondroitin sulfate proteoglycan. Some small molecular weight material indicative of free glycosaminoglycan chains was also associated with the cell layer fraction. Carbohydrate analysis of the proteoglycans demonstrated the glycosaminoglycan chains to have approximate average molecular weights of 25,000. In addition, N- and O-linked oligosaccharides which were associated with the proteoglycans appeared to be sulfated in varying degrees.     

Characterization of proteoglycans from adult bovine tendon

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.     

Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstr?m, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.     

Polydispersity and heterogeneity of squid cranial cartilage proteoglycans as assessed by immunochemical methods and electron microscopy

The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.     

Heparan sulfate proteoglycans of human neuroblastoma cells: Affinity fractionation on columns of platelet factor-4+

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca²⁺ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C₁₈ cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of “singlechain” heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.     

Proteoglycans and pattern formation: sugar biochemistry meets developmental genetics

While it has been long appreciated that sugar-modified proteins coat the cell surface, their functions are poorly understood. Here, I describe recent genetic studies that demonstrate that one class of sugar-modified proteins, cell-surface proteoglycans, play crucial roles in morphogenesis, growth regulation and tumor suppression. Mutations that affect individual proteoglycans or the enzymes required for glycosaminoglycan synthesis regulate Wingless and Decapentaplegic signaling in Drosophila, and body size in mice and humans. Compromising proteoglycan function is also associated with the development of Wilm's tumors and hereditary multiple exostoses. In this review, these biological findings are placed in the context of proteoglycan biochemistry and molecular function.     

Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture.

We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.     

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Heparan sulfate-chondroitin sulfate hybrid proteoglycan of the cell surface and basement membrane of mouse mammary epithelial cells

Chondroitin sulfate represents approximately 15% of the 35SO4-labeled glycosaminoglycans carried by the proteoglycans of the cell surface and of the basolateral secretions of normal mouse mammary epithelial cells in culture. Evidence is provided that these chondroitin sulfate-carrying proteoglycans are hybrid proteoglycans, carrying both chondroitin sulfate and heparan sulfate chains. Complete N-desulfation but limited O-desulfation, by treatment with dimethyl sulfoxide, of the proteoglycans decreased the anionic charge of the chondroitin sulfate-carrying proteoglycans to a greater extent than it decreased the charge of their constituent chondroitin sulfate chains. Partial depolymerization of the heparan sulfate residues of the proteoglycans with nitrous acid or with heparin lyase also reduced the effective molecular radius of the chondroitin sulfate-carrying proteoglycans. The effect of heparin lyase on the chondroitin sulfate-carrying proteoglycans was prevented by treating the proteoglycan fractions with dimethyl sulfoxide, while the effect of nitrous acid on the dimethyl sulfoxide-treated proteoglycans was prevented by acetylation. This occurrence of heparan sulfate-chondroitin sulfate hybrid proteoglycans suggests that the substitution of core proteins by heparan sulfate or chondroitin sulfate chains may not solely be determined by the specific routing of these proteins through distinct chondroitin sulfate and heparan sulfate synthesizing mechanisms. Moreover, regional and temporal changes in pericellular glycosaminoglycan compositions might be due to variable postsynthetic modification of a single gene product.     

Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Comparison of small proteoglycans from skin fibroblasts and vascular smooth-muscle cells.

Physicochemical and chemical properties of small proteoglycans containing galactosaminoglycan chains from cultured human skin fibroblasts and human smooth-muscle cells were compared to determine the extent of structural similarity. The proteoglycan secreted by smooth-muscle cells was of larger molecular size and of higher buoyant density, due to longer glycosaminoglycan chains, than the secretion product of skin fibroblasts. Additionally, both proteoglycans differed in the ratio of iduronic acid and glucuronic acid residues. On the other hand, degradation of secreted [3H]leucine-labelled proteoglycans with chondroitin ABC lyase followed by SDS/polyacrylamide-gel electrophoresis resulted in the appearance of core protein bands of identical size (Mr 48,000 and 45,000, depending on the number of asparagine-bound oligosaccharides). An Mr value of 40,000 was determined for the core protein of cells pretreated with tunicamycin. An antibody against the core protein from fibroblast secretions was cross-reactive with the core protein from smooth-muscle cells. Core protein accumulating intracellularly after treatment with carbonyl cyanide m-chlorophenylhydrazone exhibited, on reduction and alkylation, an isoelectric point of 7.8 in both cell types. Limited proteolysis by staphylococcal V8 serine proteinase or endoproteinase Lys-C led in both instances to the formation of peptides of identical size. Peptides bearing asparagine-bound oligosaccharides were free of glycosaminoglycan chains. Similar peptide patterns were obtained when 125I-labelled core proteins were digested with either trypsin or chymotrypsin. Thus small proteoglycans from fibroblasts and smooth-muscle cells can be differentiated by their glycosaminoglycan moieties but not by the nature of their core proteins.