A seventh member of the human 4-galactosyltransferase family, 4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of 4Gal-T7 depicts a type II transmembrane protein with sequence similarity to 4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the 4Gal-T family. The genomic organization of 4Gal-T7 was different from previous 4Gal-Ts. Expression of 4Gal-T7 in insect cells showed that the gene product had 1,4-galactosyltransferase activity with -xylosides, and the linkage formed was Gal1-4Xyl. Thus, 4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein 1,4-galactosyltransferase; EC 2.4. Six members of a family of human UDP-galactose:-Nacetylglucosamine/-glucosylceramide 1,4-galactosyltransferases (4Gal-Ts) 1 have previously been characterized (1-7).These six 4Gal-Ts catalyze biosynthesis of Gal1-4GlcNAc and/or Gal1-4Glc linkages in different glycoconjugates and free saccharides (for a review see Ref. 8). The six 4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4, 7, 9, 10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence. Detailed analysis of the kinetic properties of these enzymes clearly show that each has a distinct function in biosynthesis of different glycoconjugates and saccharide structures, but in accordance with their close evolutionary relationships the linkages formed are similar. In the present study, a seventh homologue of the 4Gal-T gene family was characterized. The gene was identified by sequence analysis of the EST data base. The coding region of the novel gene, designated 4Gal-T7, exhibited distinct substitutions in the sequence motifs highly conserved among 4Gal-T1 to 4Gal-T6. Notably, none of the four cysteines conserved among other 4Gal-Ts were found in the 4Gal-T7 sequence. It was predicted that the enzymatic properties of 4Gal-T7 were different from other 4Gal-Ts. Analysis of the substrate specificity of recombinant 4Gal-T7 revealed that this enzyme formed the Gal1-4Xyl1-R linkage found in the linkage region of proteoglycans (GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser). 4Gal-T7 was proposed to encode a galactosyltransferase I (xylosylprotein 1,4-galactosyltransferase; EC 2.4.1.133) gene (11)(12)(13). 15) showed that partial inactivation of galactosyltransferase I represented the primary defect in one patient with progeroidal appearance and symptoms of the Ehlers-Danlos syndrome. As a consequence of the enzyme deficiency, only about half of the core proteins of the small proteoglycans decorin and biglycan were linked with glycosaminoglycan chains (16), 2 whereas no abnormality in the biosynthesis of large dermatan sulf...