Recombinant Production and Characterization of Human Enzymes Responsible for Collagen Lysine Post-translational Modifications

: Biosynthesis of collagen molecules, as well as collagenous segments of non-collagen proteins, requires extensive post-translational modifications (PTMs) that confer specific functional and structural properties to tissues and organs. Within collagen polypeptide sequences, lysine residues are subject to a series of PTMs, resulting in hydroxylation and subsequent O-linked glycosylation of their side chains. These modifications are catalyzed by two distinct metalloenzyme families, named LH/PLOD and GLT25D/COLGALT, which alternatively process modified lysine side chains yielding 5-hydroxylysine (LH/PLOD), β-(1,O)-galactosyl-5-hydroxylysine (GLT25D/COLGALT), and α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine (LH/PLOD). In this chapter, we illustrate strategies to recombinantly produce human full-length LH3/PLOD3 as well as GLT25D1/COLGALT1, verify the quality of these recombinant preparations, and perform direct and indirect assays to evaluate their enzymatic activities in vitro on synthetic collagen peptides and gelatin.