GLT25D1-mediated Glycosylation of Bone Type I Collagen: Its Potential Functions

Introduction: Collagen glycosylation occurs at specific hydroxylysine (Hyl) residues producing galactosyl-Hyl (G-Hyl) and glucosylgalactosyl-Hyl (GG-Hyl). The glycosylation pattern is tissue specific and its alteration has been implicated in bone disorders. However, the molecular mechanism of this modification is still not well understood. Recently, we identified a novel glycosyltransferase 25 domain 1 (Glt25d1) expressed in mouse osteoblastic cell line, MC3T3-E1 (MC). Objective: To elucidate the role of Glt25d1 in bone type I collagen modification by employing a loss-of-function approach in vitro. Methods: MC-derived clones stably suppressing Glt25d1 (Sh clones) were generated by short-hairpin RNA technology. Type I collagen was purified from the Sh clones and from two controls, MC and those transfected with an empty vector. The site-specific glycosylation pattern of Sh type I collagen was characterized and compared with that of controls by mass spectrometry (MS). Aliquots of matrices were reduced with NaB³H₄ and subjected to quantitative cross-link analysis. Collagen fibrils in the cultures were also analyzed by transmission electron microscopy. Results: Single cell-derived clones stably suppressing Glt25d1 (by ~90-95%) were successfully generated. Five glycosylation sites were identified by MS with the predominant site found as the cross-linking site. In Sh collagen, Hyl glycosylation (GG-Hyl + G-Hyl) was found significantly decreased, with a concomitant increase in non-glycosylated Hyl.  Cross-link analysis showed that, in Sh clones, dihydroxylisinonorleucine (DHLNL) was slightly decreased with a significant increase in its maturational product, pyridinoline (Pyr). Mean collagen fibril diameters in Sh clones were significantly smaller than controls, and the diameters tended to inversely correlate to the Pyr/DHLNL ratio.  Conclusion: Glt25d1 catalyzes galactosylation of Hyl in bone type I collagen. Hyl glycosylation is a negative regulator of collagen cross-link maturation, which may in turn affect collagen fibril growth.