Abstract
Collagen is an extracellular matrix protein conserved across animals and viruses, with its function regulated by post-translational modifications of lysine residues. Specifically, certain lysine residues in collagen are hydroxylated to form hydroxylysine, which serves as an attachment site for hydroxylysine-linked glycosylation. This glycosylation process is initiated by collagen galactosyltransferases from the GT25 family, also known as GLT25D or COLGALT proteins. Despite their biological importance, efficient methods for expressing and isolating GLT25Ds have yet to be fully developed, and the biochemical mechanisms underlying their function still need to be better understood. To address this, we performed sequence alignment and phylogenetic analyses of GLT25Ds across vertebrates, invertebrates, and viruses. Using sponge (amphimedon queenslandica) GLT25D as a model, we established a bacterial expression, purification, and assay protocol. Sponge GLT25D expressed robustly in E. coli strain BL21 and demonstrated enzymatic activity comparable to human GLT25D1 from mammalian cells. Kinetic parameters and the effects of time, temperature and pH on enzymatic activity were characterized for both enzymes. AlphaFold structural modeling and sequence alignment revealed an EXD motif and a conserved leucine in a pocket of the second Rossmann-fold domain of sponge GLT25D, suggesting this pocket as the active site. Using the standardized bacterial expression, purification, and assay protocol, we screened GLT25Ds from various vertebrate and invertebrate species. Notably, the sea walnut (mnemiopsis leidyi) GLT25D exhibited superior expression levels and robust enzymatic activity. This established method provides a strong foundation for future bioengineering efforts, structure-function analyses, and the development of GLT25D inhibitors.