Raw mass spectrometry data for "Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes"

Abstract: Lysyl hydroxylase 2 (LH2) is a member of LH family of enzymes (LH1-3) that catalyze the hydroxylation
of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in Bruck syndrome,
fibrosis and cancer metastasis. Previously, we proposed LH2 as a telopeptidyl LH for type I collagen and
this specific function is now generally accepted. However, several fundamental questions remain
unanswered: 1, Is LH2 responsible for both N- (α1 and 2 chains) and C-telopeptidyl (α1 chain) Lys
hydroxylation? 2, Is LH2 involved in the helical Lys hydroxylation? 3, what are the functional
consequences when LH2 is completely lacking? To answer these questions, we generated LH2-null MC3T3
cells (LH2 KO) using CRISPR/Cas9, and extensively characterized the molecular and fibrillar phenotypes
of type I collagen. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived crosslinks
were completely absent from LH2 KO collagen with concomitant increases in the Lysald-derived crosslinks.
Mass spectrometric analysis revealed that, in LH2 KO type I collagen, telopeptidyl Lys hydroxylation
was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a sitespecific
manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl.
Furthermore, prolyl 3-hydroxylation was slightly increased in LH2 KO type I collagen. In LH2 KO samples,
collagen solubility was markedly increased, fibril diameters are significantly diminished, and
mineralization severely impaired. Together, these data underscore the critical role of LH2-catalyzed
collagen modifications and consequent cross-linking in matrix stability, organization and mineralization.