A role for disulphide bridges in the protein core in the interaction of proteodermatan sulphate and collagen.

Proteodermatan sulphate from bovine skin retarded precipitation of fibrils from solutions of purified acid-soluble bovine skin collagen. The isolated protein core was as effective as the intact proteoglycan. Thermal denaturation leading to almost complete loss of the native secondary structure, (determined by circular dichroism spectroscopy to consist of about 60% beta structure) did not diminish the effect unless accompanied by reduction of disulphides, of which there were shown to be three per molecule. The reduced and alkylated protein core was totally ineffective. Electron-microscopy revealed a D-periodic arrangement of glycosaminoglycan on the surfaces of collagen fibrils precipitated in the presence of proteodermatan sulphate. Dermatan sulphate (with attached small peptide) prepared from the proteoglycan, had no effect on the rate of fibrillogenesis and was apparently not bound to the fibrils.

Chemical and immunochemical characteristics of proteoglycans in bovine gingiva and dental pulp.

Proteoglycans were extracted with 4 M guanidinium chloride at 6 degrees C and purified by ion-exchange chromatography and precipitation with cetyl-pyridinium chloride. Chromatography on Sepharose CL-4B under dissociating conditions separated larger (PG1) and smaller (PG2) proteoglycans. Gingival PG2, by virtue of its amino-acid composition and the exclusive presence of L-iduronate-rich dermatan sulphate, was a proteodermatan sulphate (PDS) with a similar molecular weight to periodontal-ligament PDS. Reaction with four monoclonal antibodies to bovine skin PDS confirmed the relationship between these small proteoglycans and that of skin. Their glycoprotein cores, liberated by digestion with chondroitinase ABC, were similar in size (mol. wt = 55,000 by SDS-gel electrophoresis). Pulp PG2 had a small amount of PDS but the main component contained D-glucuronate-rich sulphated galactosaminoglycans. Similar galactosaminoglycans, which included chondroitin sulphate, characterized the larger proteoglycans of gingiva and pulp; significant amounts of L-iduronic acid-rich dermatan sulphate or heparan sulphate were not present.

Dermatan sulphate is located on serine-4 of bovine skin proteodermatan sulphate. Demonstration that most molecules possess only one glycosaminoglycan chain and comparison of amino acid sequences around glycosylation sites in different proteoglycans.

Digestions of bovine skin proteodermatan sulphate with cathepsin C proved that the dermatan sulphate was located on Ser-4 in most of the molecules. A Ser-Gly sequence is essential for xylosylation of the serine residue and sulphated galactosaminoglycan synthesis in different proteoglycans. Variations in adjoining sequences may be significant in relation to the glycosylation process in different tissues.

Production and characterization of monoclonal antibodies to bovine skin proteodermatan sulfate.

To study the molecular structure and function of bovine skin proteodermatan sulfate, on a determinant by determinant basis, several monoclonal antibodies to this molecule have been produced and characterized. Based on the results of a preliminary immunogenetic analysis of 4 inbred mouse strains, SJL/J (H-2s) mice were immunized for the fusions. Ten hybridomas were produced and the monoclonal antibodies from four of these were selected for further investigation. Employing an ELISA inhibition assay, none showed any detectable affinity for bovine collagen types I, II, III, or IV, bovine fibronectin or chondroitin or dermatan sulfate glycosaminoglycans. Each monoclonal antibody bound the chondroitinase ABC-derived protein core and none was significantly inhibited by proteinase digests of the intact molecule suggesting that the epitope of each contains a protein component. The results of competitive binding ELISA assays and immunoblots of the cyanogen bromide cleavage products of proteodermatan sulfate indicate that the 4 antibodies recognize at least 3 distinct antigenic determinants on this molecule. Immunohistochemical methods located the antigen in the dermis of bovine skin and revealed that a change in proteodermatan sulfate distribution occurs during skin development.

The NH2-terminal amino acid sequence of bovine skin proteodermatan sulfate.

Deglycosylation of bovine skin proteodermatan sulfate with chondroitinase ABC yielded a protein core with an apparent molecular weight of about 45,000. The amino acid sequence of this preparation was determined up to position 24. This region was enriched in acidic amino acids and proline compared with the whole protein core and it was predicted to be highly folded. The amino acid sequence determined in these experiments has a gap at position 4. Results obtained after beta-elimination-sulfite addition showed that residue 4 was an O-substituted hydroxyamino acid. The latter was identified as serine by sequencing the NH2-terminal region of the protein core (Mr approximately 43,000) isolated after a more complete deglycosylation of the proteoglycan with anhydrous HF. Serine 4 may be an attachment site for one of the few dermatan sulfate chains present in the proteoglycan.

Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans.

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.

Sulfated galactosaminoglycans of bovine periodontal ligament. Evidence for the presence of two major types of hybrids but no chondroitin sulfate.

Sulfated galactosaminoglycans of mature bovine periodontal ligament were separated into four fractions by ethanol precipitation. Fractions I and II were dermatan sulfates with high contents of L-iduronate, but only small amounts of this hexuronic acid were present in fractions III and IV. Effects of digestion with testicular hyaluronidase or a periodate-alkali treatment showed that most if not all of the glycans in fractions I, II and III were hybrid chains containing both L-iduronate and D-glucuronate. The composition of fraction IV was less certain, but the chains strongly resembled fraction III hybrids in electrophoretic characteristics, not chondroitin sulfate. The total amount of the D-glucuronate-rich fractions III and IV in the ligament was similar to that of I plus II. In contrast, almost all of the sulfated galactosaminoglycans of mature skin were rich in L-iduronate. The more varied composition of the ligament glycosaminoglycans may be related to the mixed population of cells in this tissue.

The determination of small amounts of collagen hydroxylysyl glycosides.

Glucosylgalactosylhydroxylysine (GlcGalHyl) and galactosylhydroxylysine (GalHyl) in alkaline hydrolysates of insoluble collagens were separated on 130 x 0.6 cm and 45 x 0.6 cm columns of Chromobeads A resin (Technicon) and simultaneous analyses with ninhydrin and orcinol-H2SO4 were performed. Ninhydrin, which is the standard reagent in existing methods, gave erroneously high results with both columns due to overlapping, resistant peptides. A rapid and more accurate procedure employing the smaller column, the orcinol-H2SO4 reagent and a new internal standard was developed. The losses of the glycosides in alkaline hydrolysis, especially in the presence of collagen peptides, were much larger than had been reported previously. A shorter alkaline hydrolysis following digestion of the collagen with papain was effective and allowed more reliable corrections to be made. Purging with nitrogen reduced the losses further. Mature bovine skin collagen contained less GlcGalHyl than embryonic skin collagen whereas the differences in GalHyl were insignificant.

The composition of the insoluble collagenous matrix of bovine predentine.

Predentine obtained from bovine teeth by microdissection was extracted with EDTA and tris-NaCl solutions. The insoluble residue consisted mainly of collagen and resembled dentine collagen in overall amino acid composition. The residue differed in containing no detectable phosphoprotein, a much larger amount of collagen hexose and more non-collagenous glycoprotein, the neutral sugar composition of which was determined. Differences were also observed in the contents of reducible collagen cross-links. Half of the total phosphorus found in the predentine could not be accounted for solely by hydroxyapatite. The remainder was partly soluble and dialysable.

Bovine periodontal ligament. An invesitation of the collagen, glycosaminoglycan and insoluble glycoprotein components at different stages of tissue development.

Periodontal ligaments from unerupted, partially erupted and mature teeth were extracted with 0.15 M NaCl. The major reducible collagen cross-link in each insoluble fraction was dehydrodihydroxylysinonorleucine; the dehydroydroxylysinonorleucine contents were smaller. There was no significant difference in the quantities of these cross-links relative to collagen contents in the three speciments, but one of the precursors, hydroxyallysine, markedly decreased in the older tissue. The amino acid compositions of the trypsin-resistant insoluble fractions were generally characteristic of collagen. Analyses of separated glycopeptides revealed the presence of insoluble non-collagenous glycoproteins and collagen hexoses. The latter were lower in the mature ligament. Hyaluronic acid progressively decreased relative to chondroitin sulphate on eruption and maturation. A hyaluronidase-resistant glycosaminoglycan, probably dermatan sulphate, occurred in the NaCl-insoluble fraction of the mature ligament and in appreciable amounts in all NaCl extracts.