Purification and cDNA Cloning of a Human UDP-N-acetyl-- D-galactosamine:polypeptide N-Acetylgalactosaminyltransferase

doi:10.1074/jbc.270.41.24156

Purification and cDNA Cloning of a Human UDP-N-acetyl-- D-galactosamine:polypeptide N-Acetylgalactosaminyltransferase (*)

From the (1)Faculty of Health Sciences, School of Dentistry, University of Copenhagen, DK-2200 Copenhagen N, Denmark, the
(2) Biomembrane Institute, Seattle, Washington 98119, and the
(3) Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01, Japan

^¶ To whom correspondence should be addressed:
Dept. of Oral Diagnostics, School of Dentistry, Nørre Alle 20, DK-2200 Copenhagen N, Denmark.
Tel.: 45-3532-6835; Fax: 45-3532-6505.

Abstract

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F. L., Hollanders, T., Lehman, D. J., Thomsen, D. R., and Elhammer, Å. P.(1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.

Footnotes

↵^§ The first two authors contributed equally to this work.
↵* This work was supported by the Danish Medical Research Council, the Lundbeck Foundation, and Ingeborg Roikjer's Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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) X85018 [GenBank](GalNAc-T1) and X85019 [GenBank](GalNAc-T2).
↵¹ The abbreviations used are:

GalNAc-transferase

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase

GalNAc-T1

polypeptide GalNAc-transferase originally cloned by Homa et al.(1993)

GalNAc-T2

polypeptide GalNAc-transferase cloned in the present paper

HPLC

high performance liquid chromatography

hCG

human choriogonatotropin

PCR

polymerase chain reaction

RT

reverse transcription

BisTris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride

bp

base pairs

kb

kilobase(s).
- Received May 31, 1995.