Cloning and Expression of a Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I A SEVENTH MEMBER OF THE HUMAN β4-GALACTOSYLTRANSFERASE GENE FAMILY

Abstract A seventh member of the human β4-galactosyltransferase family, β4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of β4Gal-T7 depicts a type II transmembrane protein with sequence similarity to β4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the β4Gal-T family. The genomic organization of β4Gal-T7 was different from previous β4Gal-Ts. Expression of β4Gal-T7 in insect cells showed that the gene product had β1,4-galactosyltransferase activity with β-xylosides, and the linkage formed was Galβ1–4Xyl. Thus, β4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein β1,4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Sequence analysis of β4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Roden, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342–1346), revealed two inherited allelic variants, β4Gal-T7186D and β4Gal-T7206P, each with a single missense substitution in the putative catalytic domain of the enzyme. β4Gal-T7186D exhibited a 4-fold elevated K m for the donor substrate, whereas essentially no activity was demonstrated with β4Gal-T7206P. Molecular cloning of β4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.