Fig. 1: Functional characterization of human GLT25D1.
From: Molecular basis of collagen galactosylation by GLT25D1
A Schematic representation of the collagen modification pathway. Hydroxylysine residues formed by collagen lysyl hydroxylases (LH/PLODs) at specific sites of nascent procollagen are galactosylated by GLT25D1/2. Some of these modifications undergo further glucosylation by lysyl hydroxylases. B Size-exclusion chromatography analysis of purified human GLT25D1 reveals two distinct non-aggregate elution peaks (10.3 ml and 11.8 ml, marked by dashed lines), indicative of multiple oligomeric states of GLT25D1. Inset: SDS–PAGE analysis of the eluted fractions. C In vitro galactosyltransferase activity of GLT25D1 with various acceptor substrates: gelatin, a peptide containing hydroxylysine Hyl (sequence: SGA-Hyl-GEKGS), a peptide containing lysine K instead of hydroxylysine (SGA-K-GEKGS) and an uncoupled control lacking any acceptor substrate. Activity was assessed by monitoring the production of UDP. Data are represented as mean ± s.d. for three independent replicates, evaluated using an unpaired, two-tailed Student’s t test at a 95% confidence interval (CI), and differences are depicted as ***p < 0.001; ****p < 0.0001. The exact p-values are as follows. For the peptide with hydroxylysine substrate: UDP-Gal vs UDP-Glc (p = 0.0002). For the gelatin substrate: UDP-Gal vs UDP-Glc (p < 0.0001). Mass spectrometric analysis of reaction products generated by GLT25D1 in the presence of a peptide acceptor substrate (SGA-Hyl-GEKGS; Hyl, hydroxylysine) and different donor substrates: D UDP-galactose, E UDP-glucose, F a mixture of UDP-galactose and UDP-glucose. Top row: spectra of no-enzyme controls, showing peaks (z = 3, m/z = 279.4; z = 2, m/z = 418.7; marked with blue asterisks *) corresponding to the unmodified peptide (MW of 835 Da). Bottom row: spectra of GLT25D1 reactions, showing additional peaks (z = 3, m/z = 333.4; z = 2, m/z = 499.7; marked with pink asterisks *) only in (D) and (F) that correspond to the galactosylated peptide (MW of 997 Da). Source data are provided as a Source Data file.
