GLT25D1/COLGALT1 and GLT25D2 are beta(1-O)galactosyltransferases that initiate core glycosylation of collagen by transferring galactose to hydroxylysine residues; activity is the basis for EC 2.4.1.50 assignment, with KM ~18.7 uM for UDP-galactose.

COLGALT1 was identified in a large-scale mass-spectrometry membrane-proteome screen of NK-like YTS cells; ~60% of identified proteins were judged not to be plausible integral membrane proteins but rather transiently membrane-associated.

GLT25D1/COLGALT1 is directed to the ER lumen as a soluble protein (cleaved N-terminal signal sequence) and retained there via a C-terminal RDEL ER-retrieval signal; membrane floatation shows it is not an integral membrane protein.

GLT25D1 co-localizes with its substrate mannose-binding lectin (MBL) and with the upstream lysyl hydroxylase 3 (LH3/PLOD3) in the early secretory pathway.

Mutagenesis identified aspartate residues D166/D168 and D461/D463 as essential for collagen galactosyltransferase activity, consistent with a GT-A metal-dependent catalytic fold; KM ~29.9 uM for UDP-galactose.

CRISPR/Cas9 inactivation of GLT25D1 in SaOS-2 osteosarcoma cells decreased collagen glycosylation by up to 60%, increased collagen type I expression, and caused intracellular (ER) accumulation of collagen type I, with compensatory induction of GLT25D2; loss did not alter collagen folding or thermal stability.

GLT25D1 is the main collagen galactosyltransferase isoform; GLT25D2 loss alone had no effect on collagen secretion, and clones lacking both could not be recovered, suggesting collagen glycosylation is essential for osteosarcoma cell viability.

Biallelic COLGALT1 variants (L151R, A154P, G377R) cause brain small vessel disease 3 (BSVD3); the L151R variant abolishes galactosyltransferase activity, and COLGALT1 is implicated in collagen type IV biosynthesis.