Abstract
Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases.
Highlights
LH3 is considered the evolutionary ancestor of the LH family: this isoform is the only one capable of further processing of hydroxylysines through glycosylation, whereas other isoforms might have lost such capability during evolution[16]
Reports suggest that endoplasmic reticulum (ER) retention could be mediated via interaction with distinct ER-resident proteins: LH1 is described to be part of a macromolecular complex with SC65, P3H3 and CYPB23; while LH2 forms a complex with HSP47, FKBP65 and BiP24,25; LH3 was found colocalizing with collagen galactosyltransferases GLT25D1/226
In our peptide binding measurements, all surface plasmon resonance (SPR) profiles were characterized by very fast association and dissociation events and very weak binding affinities (Supplementary Fig. 2A)
Summary
LH3 is considered the evolutionary ancestor of the LH family: this isoform is the only one capable of further processing of hydroxylysines through glycosylation, whereas other isoforms might have lost such capability during evolution[16]. Biochemical data suggest that these different enzymatic activities are localized in distinct compartments of the enzyme[19], but despite the extensive evidence available, the current knowledge of LH enzymes is far from exhaustive These enzymes are known to act together with prolyl hydroxylases, respectively introducing hydroxylations of lysine and proline residues on procollagens in the endoplasmic reticulum (ER), prior to the formation of triple-helical assemblies[20]. Extracellular LHs were reported to be active, suggesting implications for ECM stability and remodeling[27] These data indicate that lysine modifications are known to occur in the ER prior to collagen triple helical formation, secreted variants of LH3 and LH2 can modify collagens in different compartments and, possibly, in different folding states[32]. Our results offer new insights into the complex mechanisms of collagen biosynthesis and homeostasis, and provide structural templates for the development of targeted therapies for LH-related diseases and cancer
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