Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro

Type II collagen is the main structural component of hyaline cartilages whe re it forms networks of thin fibrils that differ in morphology from the muc h thicker fibrils of type I collagen. We studied here in vitro the formatio n of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low h ydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydr oxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the fo rmer was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical co ncentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggrega tion, whereas the low hydroxylysine collagen formed thick fibrils on a back ground of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major ro le in the regulation of collagen fibril formation and the morphology of the fibrils.