Molecular cloning of a human cDNA encoding β-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc β-1,4-galactosyltransferase

Molecular cloning of a human cDNA encoding β-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc β-1,4-galactosyltransferase

^*Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173, Japan; and ^§Department of Medical Chemistry, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Edited by William J. Lennarz, State University of New York, Stony Brook, NY, and approved November 18, 1997 (received for review July 10, 1997)

Abstract

A cDNA encoding a β-1,4-galactosyltransferase named β-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3′-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian β-1,4-galactosyltransferases and Lymnaea stagnalis β-1,4-N-acetylglucosaminyltransferase (β-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human β-1,4-galactosyltransferase (referred to as β-1,4-GalT I) and of 28% with that of L. stagnalis β-1,4-GlcNAcT. Study of the properties of the β-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine β-1,4-GalT but has no lactose synthetase activity in the presence of α-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the β-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another β-1,4-GalT.

Footnotes

↵ † K.F., D.H.V.D.E., and I.V.D. hold equal senior authorship in the present study.
↵ ‡ To whom reprint requests should be addressed.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviation: RACE, rapid amplification of cDNA ends.
Data deposition: The nucleotide sequence reported in this paper has been deposited in the DDBJ/GenBank/EMBL database (accession no. AB004550).