Molecular characterization of collagen lysine post-translational modification enzymes

Collagen lysine residues may undergo multiple post-translational modifications: firstly, hydroxylation mediated by LH/PLOD enzymes introduces a 5-OH moiety on the amino acid side chain. Three distinct LH/PLOD isoforms act on different lysine residues: LH2/PLOD2 is a specific telopeptide hydroxylase, whereas LH1/PLOD1 and LH3/PLOD3 act on lysine residues located within the collagen triple-helical segments. Hydroxylysines (Hyl) can undergo further galactosylation (generating Gal-Hyl), through a process mediated by GLT25D transferases. Additional glycosylation of Gal-Hyl is mediated by LH/PLOD enzymes through their N-terminal glycosyltransferase domains (generating the final Glc-Gal-Hyl product). Here, we present new results on the structural and functional characterizations of full-length human LH/PLOD and GLT25D enzymes. Structural characterizations revealed unexpected features impacting on the processivity and molecular recognition abilities of the different enzyme isoforms, as well as opportunities to map the molecular significance of pathogenic mutations affecting the genes encoding for these enzymes. Biochemical characterizations and site-directed mutagenesis rationalize the structural insights and offer new opportunities for the detailed investigation of the Lys_to_Glc-Gal-Hyl pathway in collagen biosynthesis.