Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids

doi:10.1074/jbc.274.17.11479

Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids*

From the ^‡Department of Biochemistry, Nagoya University School of Medicine, Tsurumai, Nagoya 466-0065, the ^§Department of Pediatric Dentistry, Nagasaki University School of Dentistry, Sakamoto, Nagasaki 852-8501, and the ^¶Department of Applied Bio-organic Chemistry, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Abstract

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Footnotes

↵* This work was supported by Grant-in-aid for Scientific Research in Priority Areas 10178105, by a Core of Excellence grant from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant-in-aid from Ono Medical Research Foundation.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) AB022918.
↵‖ To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466 Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.
↵2 Fukumoto, S., Miyazaki, H., Urano, T., Furukawa, K., and Furukawa, K. (1999) J. Biol. Chem. 274, in press.
Abbreviations:

CMP-NeuAc

cytidine 5′-monophospho-N-acetylneuraminic acid

mAb

monoclonal antibody

RT

reverse transcription

PCR

polymerase chain reaction

DMEM

Dulbecco’s modified Eagle’s medium

FCS

fetal calf serum

PBS

phosphate-buffered saline

TLC

thin layer chromatography

PAGE

polyacrylamide gel electrophoresis

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HPTLC

high performance thin layer chromatography

kb

kilobase pair(s)

SPG

sialyl-paragloboside. The nomenclature of gangliosides is based on that of Svennerholm (54). The abbreviated nomenclature for cloned sialyltransferases follows Ref.24. The designations of glycosphingolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Nomenclature (55). Lc4, Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ1-Cer

nLc4

Galβ1,4GlcNAcβ1,3Galβ1,4Glcβ1-Cer

nLc6

Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Gal β1,4Glcβ1-Cer

Le^x

Lewis X (Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glcβ1-Cer)
- Received November 18, 1998.
- Revision received February 4, 1999.
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