Specificity of the low density lipoprotein-glycosaminoglycan interaction.

There is ample documentation of the binding of chondroitin sulfate/dermatan sulfate proteoglycans to low density lipoprotein (LDL) both in vivo and in vitro. The interaction of these two species may be an early and important step in atherogenesis. Therefore, there is interest in defining the features of both molecules that are critical for their interaction. We employed a recently described competitive microassay that measures initial binding of proteoglycan to immobilized LDL. We confirmed the work of others that it is the apolipoprotein B component and, at least in part, a heparin-binding domain of LDL that are responsible for binding chondroitin sulfate/dermatan sulfate proteoglycans. The principal thrust of our study was concerned with the effects of a glycosaminoglycan's degree of sulfation on the binding to LDL. Initial experiments comparing dermatan sulfate and chondroitin sulfate proteoglycans indicated that the former was more efficient at binding LDL than the latter and that oversulfation, rather than chain length or iduronate content, was the preeminent feature involved. Additional binding studies with dermatan sulfate, chemically sulfated chondroitin-4-sulfate, and naturally occurring oversulfated chondroitin sulfates indicated that the degree of sulfation, not the position of sulfation, determined affinity for LDL. These results suggest that studies should be undertaken to determine whether oversulfated segments of glycosaminoglycans are especially involved in associations with LDL, leading to lipid accumulation, in the artery wall.

An unsulphated region of the rat chondrosarcoma chondroitin sulphate chain and its binding to monoclonal antibody 3B3.

The chondroitin sulphate chains of proteoglycans are not uniformly sulphated. Commonly, regions of under- and over-sulphation are found. It is probable that variability in chondroitin sulphation has physiological significance, although such structure-function relationships largely remain unexplored. Chondroitin sulphate from rat chondrosarcoma proteoglycan has been found to possess no oversulphated residues. It is primarily chondroitin 4-sulphate, although a significant proportion of unsulphated disaccharides (14%) are also present. It appears that some unsulphated disaccharides are concentrated only at the point of attachment to the linkage region (i.e. it is the major unsaturated disaccharide remaining attached to chondrosarcoma proteoglycan core produced by chondroitinase ABC digestion). This proteoglycan core binds monoclonal antibody (MAb) 3B3. Although 3B3 principally binds to 6-sulphated 'stubs' of proteoglycan cores [Couchman, Caterson, Christner & Baker (1984) Nature (London) 307, 650-652], given a high concentration of unsulphated 'stubs', it can alternatively bind to these residues. It is also evident that caution must be exercised in using MAb 3B3 to identify chondroitin 6-sulphated proteoglycans.

A competitive assay of lipoprotein: proteoglycan interaction using a 96-well microtitration plate.

A method for the microassay in vitro of lipoprotein: proteoglycan interactions is described. The wells of a plastic 96-well microtitration plate are coated with low density lipoprotein. A limiting quantity of biotin-conjugated proteoglycan is allowed to bind to each coated well, and the amount of the latter retained in wells is estimated spectrophotometrically through subsequent binding of alkaline phosphatase-conjugated avidin. Many of the incubation parameters (e.g., time, pH, salt concentration, divalent cations), which influence the extent of binding of biotin-conjugated proteoglycan, have been studied and optimized. The effect upon binding of introducing different levels of proteoglycans or lipoproteins at the interaction step can be measured readily. Thus, the orders of increasing relative binding affinities were found to be high density lipoprotein less than Lipoprotein (a) less than low density lipoprotein; rat chondrosarcoma proteoglycan less than bovine nasal cartilage proteoglycan less than human aorta proteoglycan; chondroitin 4-sulfate less than chondroitin 6-sulfate less than dermatan sulfate for lipoproteins, proteoglycans, and glycosaminoglycans, respectively.

Biosynthesis of chondroitin sulfate proteoglycan by P388D1 macrophage-like cell line.

Macrophages are in large part responsible for the subendothelial deposition of lipid within the artery wall during the early stages of atherogenesis. Proteoglycans secreted by these cells may play a role in this pathological process either by trapping lipoproteins in the extracellular matrix or by enhancing the formation of lipid-laden foam cells. The synthesis and secretion of proteoglycan was studied in the P388D1 macrophage-like cell line cultured in the presence of 35S-sulfate. The radiolabeled proteoglycan had a Kd of 0.69 on Sepharose CL-2B corresponding to an Mr of 2.8 x 10(5). It consisted of approximately 13 chondroitin sulfate chains of Mr 20,000 attached to a core protein with an Mr of 18,000. The chondroitin sulfate chains contained both N-acetylgalactosamine 6-sulfate and N-acetylgalactosamine 4-sulfate residues. No disulfated N-acetylgalactosamine residues were present. The P388D1 proteoglycan bound specifically to immobilized human low density lipoprotein. These results suggest that, in the focal regions of the arterial wall in which macrophages are found during the development of fatty streaks, proteoglycans secreted by these cells may affect the transport and cellular metabolism of plasma-derived lipids.

Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures.

Cartilage proteoglycan aggregates contain two components (proteoglycan monomer and link protein) which interact with each other and with hyaluronic acid. Data from amino acid sequence analysis are presented that shows that a domain of the proteoglycan, the hyaluronic acid binding region, which interacts with link protein and hyaluronic acid is very similar to link protein in terms of its primary structure. However, the pattern of glycosylation in the hyaluronic acid binding region is different from that found in link protein. After removal of N-linked oligosaccharides, the tryptically prepared hyaluronic acid binding region from rat chondrosarcoma has a mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 43 +/- 2 kDa. The COOH-terminal two-thirds of rat chondrosarcoma link protein, starting at residue 105, has 41.3% identity with a similar region in the hyaluronic acid binding region. We show that, in addition to the hyaluronic acid binding region, proteoglycan contains another region with similarity to the two repeating loop structures in the COOH-terminal two-thirds of link protein. This presumably corresponds to the second globular domain reported in rotary shadowing studies of cartilage proteoglycans. We have deduced the positions of all of the disulfide bonds in the hyaluronic acid binding region and find them to be in the same positions as would be expected from comparison of these sequences with link protein.

The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate.

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, a glycoprotein interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. The primary structure of the link protein has been determined with a view to defining its interaction with both hyaluronic acid and proteoglycan. Thus, the link protein has been digested with staphylococcal V8 protease, trypsin, and chymotrypsin and the resulting peptides characterized by amino acid composition and sequence. We have determined that the link protein is a single peptide with 339 amino acid residues. The protein core has a molecular weight of 38,564. There is one N-linked oligosaccharide at residue 41 with a molecular weight of approximately 2,500. There are five disulfide bonds which define three loops within the amino acid sequence. The loop nearest to the NH2-terminal contains 78 amino acids and is followed by a section of 42 amino acids between it and the second loop. The second and third loops display considerable homology with each other; they consist of 71 and 70 amino acids, respectively, each contain two disulfide bonds, and both loops possess, approximately centrally, an epitope for the species nonspecific anti-link protein monoclonal antibody, 8A4. These loops are separated by a short section of 27 amino acids. We speculate that these loops are functionally important in the interaction of link protein with hyaluronic acid, as they appear to be the most conserved regions of link protein between species.

Articular cartilage proteoglycans from normal and osteoarthritic mice.

Articular cartilage proteoglycans from an osteoarthritic mouse strain, STR/IN, were labeled in vivo with 35S-sulfate and characterized with respect to extractability, ability to aggregate, size of monomer and glycosaminoglycan chains, sulfation of glycosaminoglycans, relative amounts of chondroitin-4 sulfate and chondroitin-6 sulfate, and link proteins. The proportion of 35S-labeled proteoglycans extractable by 0.4M guanidine hydrochloride was the same in control and osteoarthritic animals. However, a greater proportion was extractable by 4M guanidine hydrochloride in the STR/IN animals as compared with the control mice. The ability of the 35S-proteoglycans to aggregate was comparable in controls and osteoarthritic mice, as judged by their exclusion on Sepharose CL-2B. Monomers from both controls and osteoarthritic animals eluted from Sepharose CL-2B with a KAV of 0.47. Glycosaminoglycans from control and osteoarthritic animals eluted from Sepharose CL-6B with a KAV of 0.63, and no differences in sulfation or chondroitin-4 sulfate content were found. Aggregates were immunoprecipitated with link protein-specific antiserum, and only link protein 2 was found in aggregates from control and osteoarthritic animals.

Characterization of a dermatan sulfate proteoglycan synthesized by murine parietal yolk sac (PYS-2) cells.

A dermatan sulfate proteoglycan has been isolated from a murine parietal yolk sac cell line, which in culture synthesizes basement membrane components. The proteoglycan has a molecular weight of 200,000-300,000 with 10-15 dermatan sulfate chains of Mr = 14,000-16,000. The glycosaminoglycan chains carry sulfate residues predominantly attached to C-4 of the galactosamine unit; less than 10% of the sulfate groups occur as 6-sulfated galactosamine units. About 60% of the uronic acid residues are of the glucuronic configuration, the rest being iduronic acid. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chondroitinase ABC-treated 125I-labeled proteoglycan reveals two polypeptides with molecular weights of 34,000 and 27,000. Results from papain digestion of the proteoglycan suggest that most of the polysaccharide chains are clustered at a papain-resistant segment of the core protein (Mr = 8,000). This proteoglycan is distinctly different from the large cartilage proteoglycan in the smaller size of its core protein, and its relationship to other small chondroitin and dermatan sulfate proteoglycans and to the chondroitin sulfate proteoglycan recently located in rat tissue basement membranes will be discussed.

An amino acid sequence common to both cartilage proteoglycan and link protein.

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. In the course of determining the primary structure of link protein, two peptides produced by digestion of rat chondrosarcoma link protein with trypsin or chymotrypsin have been selectively purified by immunoaffinity chromatography on a column of monoclonal anti-link protein antibody (8A4) immobilized to Sepharose 4B. These peptides have been sequenced using the double-coupling dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate procedure. A consensus sequence, Cys-X-Ala-Gly-Trp-Leu-X-Asp-Gly-Ser-Val-X-Tyr-Pro-Ile-X-X-Pro, obtained by comparing the affinity-isolated tryptic peptide with the affinity-isolated chymotryptic peptide and an overlapping tryptic peptide, shows homology with a sequence obtained from the NH2-terminal of a CNBr peptide from proteo glycan core protein of bovine nasal cartilage: Ser-Ser-Ala-Gly-Trp-Leu-Ala-Asp-Arg-Ser-Val-Arg-Tyr-Pro-Ile-Ser-. We suggest that the common sequence is structurally important to the function of these proteins and may be involved in the binding of both link protein and proteoglycan to hyaluronic acid.

Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan.

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans.

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.

Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues.

Chondroitin sulphate proteoglycans are widespread connective tissue components and chemical analysis of cartilage and other proteoglycans has demonstrated molecular speciation involving the degree and position of sulphation of the carbohydrate chains. This may, in turn, affect the properties of the glycosaminoglycan (GAG), particularly with respect to self-association and interactions with other extracellular matrix components. Interactions with specific molecules from different connective tissue types, such as the collagens and their associated glycoproteins, could be favoured by particular charge organizations on the GAG molecule endowed by the sulphate groups. So far, it has not been possible to identify and map chondroitins of differing sulphation in tissues, but we have now raised three monoclonal antibodies which specifically recognize unsulphated, 4-sulphated and 6-sulphated chondroitin and dermatan sulphate. These provide novel opportunities to study the in vivo distribution of chondroitin sulphate proteoglycans. We demonstrate that chondroitin sulphates exhibit remarkable connective tissue specificity and furthermore provide evidence that some proteoglycans may predominantly carry only one type of chondroitin sulphate chain.

Proteoglycans in basement membranes.

Previous studies have shown that sulphated proteoglycans are integral components of basement membranes. We have used mouse parietal yolk sac cells as a model system for our studies. These cells produce several basement membrane components, including a heparan sulphate proteoglycan and a chondroitin sulphate proteoglycan. The structure of the heparan sulphate proteoglycan has been described previously. The chondroitin sulphate proteoglycan has an Mr of 200 000-300 000 and contains 10-20 chondroitin sulphate chains (Mr = 14 000-16 000), attached to a core protein that on polyacrylamide gel electrophoresis appears as a doublet (with Mr = 34 000 and 27 000). Further structural analysis suggests that the majority of the polysaccharide chains are clustered around one segment of the core protein. The polysaccharide chains carry sulphate residues predominantly attached to C-4 of the galactosamine unit. More than 60% of the uronic acid residues are of the glucuronic configuration, the rest being iduronic acid. The parietal yolk sac cells secrete about equal amounts of the two proteoglycans into the culture medium, whereas heparan sulphate proteoglycan is the predominant proteoglycan found in the extracellular matrix of these cells. This proteoglycan appears to be anchored in the matrix by interactions involving the core protein rather than the polysaccharide chains.

Studies on the properties of the inextractable proteoglycans from bovine nasal cartilage.

Bovine nasal cartilage was repeatedly extracted with the dissociative solvent, 4 M guanidinium chloride. About 25% of the total tissue proteoglycans could not be solubilized as judged by galactosamine analysis. The inextractable proteoglycan could be at least partially solubilized by treating the guanidine-extracted tissue with reagents which completely or partially degraded the proteoglycan protein core. Digestion with papain or cyanogen bromide completely solubilized the tissue and released all of the galactosamine-containing material while trypsin or hydroxylamine treatment left the cartilage macroscopically unchanged and extracted about 50% of the residual galactosamine. The degradatively solubilized material was compared to that extracted with guanidinium chloride. The papain-released glycosaminoglycan chains from the two proteoglycan preparations were similar with respect to size, degree of sulfation, position of sulfation, and hexosamine content. Furthermore, the fragments released from the cartilage residue by either cyanogen bromide, trypsin, or hydroxylamine treatment were of the same size, as judged by gel chromatography, as those derived from similarly digested guanidine-extracted proteoglycan. Trypsin digestion also released the keratan sulfate-enriched peptide as well as peptides from the hyaluronic acid-binding region. By the methods used, the inextractable proteoglycan appears to be similar to the fraction which is readily soluble under dissociative conditions and thus may be held tightly within the tissue by a nonspecific mechanism(s) such as entanglement in the collagen fibril network.

Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan.

Monoclonal antibodies were raised against proteoglycan core protein isolated after chondroitinase ABC digestion of human articular cartilage proteoglycan monomer. Characterization of one of the monoclonal antibodies (1/20/5-D-4) indicated that it specifically recognized an antigenic determinant in the polysaccharide structure of both corneal and skeletal keratan sulfate. Enzyme immunoassay analyses indicated that the mouse monoclonal IgG1 recognized keratan sulfate in native proteoglycan aggregate and proteoglycan monomer preparations isolated from hyaline cartilages of a wide variety of animal species (human, monkey, cow, sheep, chicken, and shark cartilage). The 1/20/5-D-4 monoclonal antibody did not recognize antigenic determinants on proteoglycan isolated from Swarm rat chondrosarcoma. This finding is consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue. A variety of substructures isolated after selective cleavage of bovine nasal cartilage proteoglycan (Heinegård, D., and Axelsson, J. (1977) J. Biol. Chem. 252, 1971-1979) were used as competing antigens in radioimmunoassays to characterize the specificity of the 1/20/5-D-4 immunoglobulin. Substructures derived from the keratan sulfate attachment region of the proteoglycan (keratan sulfate peptides) showed the strongest inhibition. Both corneal and skeletal keratan sulfate peptides as competing antigens in radioimmunoassays showed similar inhibition when compared on the basis of their glucosamine content. Therefore, the 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties. Chemical desulfation of the keratan sulfate reduced the antigenicity of the glycosaminoglycan. The antibody did not recognize determinants present in dermatan sulfate, heparin, heparin sulfate, or hyaluronic acid.

Immunological methods for the detection and determination of connective tissue proteoglycans.

In this paper we report the use of immunological methods for specifically detecting and determining proteoglycan in cartilage and other connective tissues. Antibodies (polyclonal and monoclonal) have been raised against specific components of cartilage proteoglycan aggregates (i.e., proteoglycan monomer and link protein). Radioimmunoassay procedures and immunohistochemical procedures have been developed and used to demonstrate the occurrence of cartilage-like proteoglycan and link protein in bovine aorta. Similarly, immunofluorescent studies have been used to analyze proteoglycan distribution in skin. Using antibodies specific for chondroitin-4-sulfated proteoglycan, their presence was demonstrated in dermal connective tissue and connective tissue surrounding nerve and muscle sheaths. However, chondroitin-4-sulfated proteoglycan was completely absent in the epidermis of skin and areas surrounding invaginating hair follicles. These immunological procedures are currently being used to complement conventional biochemical analyses of proteoglycans found in different connective tissue matrices.