Tài liệu Báo cáo Y học: Human and Drosophila UDP-galactose transporters transport

Human and Drosophila UDP-galactose transporters transport

UDP-N-acetylgalactosamine in addition to UDP-galactose

Hiroaki Segawa*, Masao Kawakita  and Nobuhiro Ishida

Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Honkomagome,

Bunkyo-ku, Tokyo, Japan

A putative Drosophila nucleotide sugar transporter was

characterized and shown to be the Drosophila homologue of

the human UDP-Gal transporter (hUGT). When the

Drosophila melanogaster UDP-Gal transporter (DmUGT)

was expressed in mammalian cells, the transporter protein

was localized in the Golgi membranes and complemented

the UDP-Gal transport de®ciency of Lec8 cells but not the

CMP-Sia transport de®ciency of Lec2 cells. DmUGT and

hUGT were expressed in Saccharomyces cerevisiae cells in

functionally active forms. Using microsomal vesicles isolated

from Saccharomyces cerevisiae expressing these transporters,

we unexpectedly found that both hUGT and DmUGT

could transport UDP-GalNAc as well as UDP-Gal. When

amino-acid residues that are conserved among human,

murine, ®ssion yeast and Drosophila UGTs, but are distinct

from corresponding ones conserved among CMP-Sia

transporters (CSTs), were substituted by those found in

CST, the mutant transporters were still active in transporting

UDP-Gal. One of these mutants in which Asn47 was sub￾stituted by Ala showed aberrant intracellular distribution

with concomitant destabilization of the protein product.

However, this mutation was suppressed by an Ile51 to Thr

second-site mutation. Both residues were localized within the

®rst transmembrane helix, suggesting that the structure of

the helix contributes to the stabilization and substrate rec￾ognition of the UGT molecule.

Keywords: UDP-galactose transporter; UDP-galactose;

UDP-N-acetylgalactosamine; nucleotide sugar transporter;

site-directed mutagenesis.

Oligosaccharide chains of secretory and membrane-bound

glycoproteins and glycolipids play important roles in

various biological processes. Two major groups of proteins,

nucleotide sugar transporters (NSTs) and glycosyltransfe￾rases, contribute to oligosaccharide synthesis. Nucleotide

sugar transporters carry speci®c nucleotide sugars that are

produced outside the Golgi apparatus and ER into these

organelles, where they serve as the substrates for the

elongation of carbohydrate chains by appropriate glyco￾syltransferases. Changes in the activities of NSTs may

affect the structure of oligosaccharide chains by affecting

the availability of substrates for glycosyltransferases [1].

In fact, in organisms such as Drosophila melanogaster and

Caenorhabditis elegans, de®ciencies in enzymes involved in

oligosaccharide biosynthesis and putative nucleotide sugar

transporters lead to abnormal development of these orga￾nisms [2,3]. However, the regulation of glycoconjugate

structure through the availability of nucleotide sugar

substrates remains unclear, because much less attention

has been paid so far to NSTs than to glycosyltransferases

and because the molecular detail of NST structures has not

been determined until quite recently.

Several NST genes have been isolated recently from

organisms including yeasts [4±7], protozoa [8], worms [9],

and mammals [10±16]. These genes encode structurally

related hydrophobic membrane proteins. The UDP-Gal

transporter (UGT), UDP-GlcNAc transporter (UGlcN￾AcT) and CMP-Sia transporter (CST) show considerable

similarity with each other, but have distinct substrate

speci®cities. The mechanisms underlying the speci®c sub￾strate recognition are intriguing, but remain obscure.

Alignment of new members of the NST family with other

family members may offer clues about the mechanisms of

substrate recognition by NSTs.

In this communication, we describe the molecular

cloning and characterization of a Drosophila homologue

of mammalian NST (DmNST), which we found in the

D. melanogaster expressed sequence tag (EST) database.

The deduced amino-acid sequence of DmNST showed

moderate similarity to hUGT, hUGlcNAcT and hCST, and

heterologous expression in yeast allowed us to identify the

Correspondence to M. Kawakita, Department of Applied Chemistry,

Kogakuin University, 1-24-2 Nishi-Shinjuku, Shinjuku-ku, Tokyo

163-8677, Japan. Fax: + 81 3 3340 0147, Tel.: + 81 3 3340 2731,

E-mail: bt13004@ns.kogakuin.ac.jp

Abbreviations: NST, nucleotide sugar transporter; UGT, UDP￾galactose transporter; UGlcNAcT, UDP-N-acetylglucosamine trans￾porter; CST, CMP-sialic acid transporter; UDP-Gal, UDP-galactose;

UDP-GlcNAc, UDP-N-acetylglucosamine; CMP-Sia, CMP-sialic

acid; UDP-GalNAc, UDP-N-acetylgalactosamine; DmNST,

Drosophila melanogaster NST; EST, expressed sequence tag; hUGT,

human UDP-galactose transporter; hCST, human CMP-sialic acid

transporter; hUGlcNAcT, human UDP-N-acetylglucosamine trans￾porter; HA, in¯uenza virus hemagglutinin; FITC, ¯uorescein

isothiocyanate; GS-II, Gri€onia simplicifolia lectin II; PNA, peanut

agglutinin.

*Present address: Department of Biochemistry, University of Ken￾tucky Medical Center, College of Medicine, Lexington, KY, USA.

 Present address: Department of Applied Chemistry, Kogakuin

University, Nishi-Shinjuku, Shinjuku-ku, Japan.

Note: the nucleotide sequence for DmUGT reported in this paper has

been submitted to the GenBank/EMBL/DDBJ under accession

number AB055493.

(Received 31 August 2001, accepted 24 October 2001)

Eur. J. Biochem. 269, 128±138 (2002) Ó FEBS 2002