Characterization and Quantitative Determination of the Hydroxylysine-linked Carbohydrate Units of Several Collagens

Abstract The hydroxylysine-containing glycopeptides from collagenase-Pronase digests of bovine tendon collagen were found to have glucose and galactose as their monosaccharide components. The ratio of galactose to glucose in these glycopeptides was greater than unity and after alkaline hydrolysis both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found. In order to determine the content of glucosylgalactosylhydroxylysine, galactosylhydroxylysine, and unsubstituted hydroxylysine present in tendon collagen as well as in several other collagens, analyses for these components were performed on the amino acid analyzer after alkaline hydrolysis. The other collagens studied were obtained from rat skin, rat tail tendon, carp swim bladder, rabbit sclera, and corneas from rabbits and calves. From most of these sources both the citrate-soluble and citrate-insoluble collagens were studied. In all of the proteins studied both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found, with the galactosylhydroxylysine making up from 46% of the total hydroxylysine-linked carbohydrate units in calf skin citrate-soluble collagen to 11% in carp swim bladder citrate-insoluble collagen. The citrate-insoluble collagens tended to contain a greater percentage of glucosylgalactosylhydroxylysine units than the citrate-soluble collagens from the same source. The percentage of the total hydroxylysine residues substituted by the glycosidic linkages of these units varied from 5% in rat tail tendon citrate-soluble collagen (0.4 carbohydrate unit/1000 amino acid residues) to 56% in the rabbit cornea citrate-insoluble collagen (5.8 carbohydrate units/1000 amino acid residues). All these collagens were shown to contain markedly fewer hydroxylysine-linked carbohydrate units than basement membranes, suggesting that higher levels of hydroxylysine-linked carbohydrate are reflected in lower degrees of morphological organization as seen under the electron microscope. The glucosylgalactosylhydroxylysine and galactosylhydroxylysine obtained from the alkaline hydrolyses of the various collagens had the same migration on paper and ion exchange chromatography as the 2-O-α-d-glucosyl-O-β-d-galactosylhydroxylysine and O-β-d-galactosylhydroxylysine previously isolated from the bovine renal glomerular basement membrane. For structural studies glycopeptides containing the disaccharide units were obtained from the collagenase-Pronase digest of ichthyocol. The preferential release of glucose during graded acid hydrolysis of these glycopeptides and the isolation of a disaccharide with galactose on the reducing end indicated a glucosylgalactose sequence in these carbohydrate units. The disaccharide was split by α-glucosidase but was resistant to the action of β-glucosidase. Periodate oxidation, galactose oxidase treatment, and methylation of the glycopeptides indicated that the glucose is linked glycosidically to carbon-2 of the galactose. These results are consistent with a structure for the disaccharide of 2-O-α-d-glucopyranosyl-d-galactose, which is identical with that found for the glomerular basement membrane. In addition to glucose and galactose, the collagens contained small amounts of other sugar components including mannose, fucose, hexosamines, and sialic acids. In the collagenase-Pronase digests of ichthyocol and tendon collagen, glycopeptides containing these sugars could be separated from most of the hydroxylysine-containing glycopeptides by gel filtration.