Bovine α1→3-Galactosyltransferase: Isolation and Characterization of a cDNA Clone

Abstract

We have isolated, by immunological screening of a lambda gt11 expression library, a cDNA clone that represents the complete coding sequence for bovine alpha 1----3-galactosyltransferase. The coding sequence predicts a membrane-bound protein with three distinct structural features: a large, potentially glycosylated COOH-terminal domain (346 amino acids), a single transmembrane domain (16 amino acids), and a short NH2-terminal domain (6 amino acids). Thus, the domain structure for this transferase is similar to that deduced for beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428) and alpha 2----6-sialyltransferase (Weinstein, J., Lee, E. V., McEntee, K., Lai, P.-H., and Paulson, J. C. (1987) J. Biol. Chem. 262, 17735-17743). S1 analysis demonstrates that two sets of mRNAs, which are heterogeneous at their 5' ends, are transcribed. Because both sets initiate upstream of the translational start site, only one protein is encoded by this gene. alpha 1----3-Galactosyltransferase is widely expressed in different mammalian species, with the notable exception of man and Old World monkeys (Galili, U., Shohet, S. B., Kobrin, E., Stults, C.L.M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762). By Northern blot analysis we were indeed unable to detect transcripts for this enzyme in various human and Old World monkey cell lines; transcripts were readily detected in other mammalian species. However, by Southern blot analysis, homologous sequences for alpha 1----3-galactosyltransferase were identified in human genomic DNA. This suggests that the gene, although present in the human genome, is normally not expressed. These observations have potential medical implications. Because many humans have high levels of circulating antibodies directed against the enzymatic product of alpha 1----3-galactosyltransferase (Gal alpha 1----3Gal beta 1----4GlcN Ac) (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373), it has been suggested that activation of this normally silent gene may play a role in autoimmune disease in man (Etienne-Decerf, J., Malaise, M., Mahieu, P., and Winand, R. (1987) Acta Endocrinol. 115, 67-74).