Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

Stefan Oelmann,Pamela Stanley,Rita Gerardy-Schahn

doi:10.1074/jbc.m011124200

Abstract

Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.

Highlights

The maturation of glycoconjugates that are either secreted or become constituents of the plasma membrane occurs while passing through the Golgi compartments
Identifying point mutations that inactivate UDP-galactose transporter (UGT) in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CMP-sialic acid transporter (CST), the two most divergent mammalian Nucleotide-sugar transporters (NSTs)
Molecular Cloning and Expression of the Hamster UGT—To isolate a hamster UGT cDNA, a cDNA library from CHO K1 cells was screened using colony hybridization with a digoxigenin-labeled probe derived from the 3Ј part of the coding region of isoform 1 of the human UGT (UGT1; Ref. 9; accession number D84454)

Summary

Introduction

The maturation of glycoconjugates that are either secreted or become constituents of the plasma membrane occurs while passing through the Golgi compartments. Three genetic complementation groups that are affected in the UDP-galactose transporter (UGT) gene have been described: the CHO mutant Lec8 [6], murine Had-1 cells [7], and a vanadium-sensitive strain of Schizosaccharomyces pombe called gms (for galactomannan synthesis-negative) [8]. Little information exists on the functional modi and architecture of the NSTs. Membrane topology has been determined for the murine CMP-sialic acid transporter (CST) and, in contrast to the theoretically predicted eight transmembrane domains, it was shown to contain 10 transmembrane domains [14]. The cytosolically located N- and C-terminal tails of murine UGT were found to be dispensable. Deletion of both ends affected neither the activity nor the subcellular destination of the UGT [11]

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