Cloning and Expression of a Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I

doi:10.1074/jbc.274.37.26165

Cloning and Expression of a Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I

A SEVENTH MEMBER OF THE HUMAN β4-GALACTOSYLTRANSFERASE GENE FAMILY*

From the ^‡School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, the ^§Institute of Molecular Pathology and Immunology of University of Porto, Rua Dr. R. Frias s/n, 4200 Porto, Portugal, the ^¶University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia 30602, and the ^‖Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48129 Münster, Germany

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., Rodén, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342–1346), revealed two inherited allelic variants, β4Gal-T7^186D and β4Gal-T7^206P, each with a single missense substitution in the putative catalytic domain of the enzyme. β4Gal-T7^186D exhibited a 4-fold elevated K _m for the donor substrate, whereas essentially no activity was demonstrated with β4Gal-T7^206P. Molecular cloning of β4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.

Footnotes

↵* This work was supported by the Danish Cancer Society, the Velux Foundation, the Danish Research Council, Praxis Grant XXI 2/2.1/BIA/276/94, National Institutes of Health Resource Center for Biomedical Complex Carbohydrates Grant 5 P41 RR05351, and Deutsche Forschungsgemeinschaft Grant SFB 310, Project B2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
↵** To whom correspondence should be addressed: School of Dentistry, Nørre Alle 20, DK-2200 Copenhagen N, Denmark. Tel.: 45-35326835; Fax: 45-35326505; E-mail: henrik.clausen@odont.ku.dk.
↵2 H. Kresse, unpublished observation.
↵3 H. Kresse, unpublished observation.
↵4 Gastinel, L. N., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 3546–3557.
↵5 R. Almeida and H. Clausen, unpublished observation.
Abbreviations:

β4Gal-T

UDP-galactose:β-N-acetylglucosamine/β-glucose/β-xylose β1,4-galactosyltransferase

EST

expressed sequence tags

MeUmb

methylumbelliferyl

TOCSY

total correlation spectroscopy

HSQC

heteronuclear single quantum correlation

HMBC

heteronuclear multiple bond correlation

PCR

polymerase chain reaction
- Received June 3, 1999.
- Revision received July 7, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.