Roles of lysyl hydroxylase-2 in collagen cross-linking, fibrillogenesis, and mineralization

Lysine (Lys) hydroxylation is one of the post-translational modifications of collagen crucial for collagen cross-linking and glycosylation. The process of Lys hydroxylation is catalyzed by lysyl hydroxylase (LH), encoding by Procollagen-lysine, 2-oxyglutarate 5-dioxygenase gene. Currently, three isoforms of LH have been identified both in human and mouse tissues but their specific functions are not well defined. The hypothesis of this study is that LH2 modulates the pattern of collagen cross-linking, fibrillogenesis and subsequent mineralization partially through its action on telopeptidyl Lys residues of the collagen molecule. To test the hypothesis, the MC3T3-E1 osteoblastic cell clones expressing higher levels (S clones) and lower levels (As clones) of the predominant form of LH2 by overexpression and antisense approaches were established and collagen cross-linking of these clones were characterized. Fibrillogenesis and mineralization produced by the S clones were investigated. In the S clones, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than that of controls and the level of pyridinoline (Pyr) was elevated. In contrast, a decrease in DHLNL/HLNL ratio, an absence of Pyr, and significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were found in the As clones. The collagen fibrils formed by the S clones were significantly smaller than those of controls. Furthermore, the mineralized matrice produced by the S clones were less or delayed, compared to those of controls. The data clearly demonstrate a critical role of LH2b in determining collagen cross-linking pathways most likely through its action on telopeptidyl Lys residues, collagen fibrillogenesis and subsequent matrix mineralization.