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

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 ⌬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.

Transport of UDP-galactose in Plants

“…Moreover, they share some consensus sequences where key amino acid residues have been shown to be essential for the transport of UDP-galactose (6). Although AtUTr1 also transports UDP-Gal, we do not find these conserved sequences in the protein.…”

“…Complementation of mutants allowed the cloning of putative UDP-galactose transporter genes from human and Schizosaccharomyces pombe (6,10). Through PCR-based approaches and screening of libraries, murine, hamster, and Drosophila cDNAs were also isolated (6,7,11). Expression of these genes in Saccharomyces cerevisiae followed by in vitro transport assays into Golgi vesicles confirmed that the gene products actually transport UDP-galactose (12).…”

“…UDP-galactose transporters have been described in animal cells, Drosophila, and yeast (3)(4)(5)(6)(7). Studies in MDCK 1 cells and yeast mutants deficient in transport of UDP-galactose into the Golgi lumen have shown that they play important roles in galactosylation of proteins, proteoglycans, and sphingolipids (8 -10).…”

“…Studies in MDCK 1 cells and yeast mutants deficient in transport of UDP-galactose into the Golgi lumen have shown that they play important roles in galactosylation of proteins, proteoglycans, and sphingolipids (8 -10). Complementation of mutants allowed the cloning of putative UDP-galactose transporter genes from human and Schizosaccharomyces pombe (6,10). Through PCR-based approaches and screening of libraries, murine, hamster, and Drosophila cDNAs were also isolated (6,7,11).…”

Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes

“…This defect in CMP-sialic acid transport results in N-and O-glycans with a greater than 90% decrease in sialic acid content (35). The Lec8 mutant has a deletion mutation in the UDP-galactose transporter resulting in a truncated protein with a greatly reduced ability to translocate UDP-Gal into the lumen of the Golgi apparatus (36). Thus, Lec8 cells generate nongalactosylated and nonsialylated N-and O-glycans (37).…”