Molecular Cloning of the Hamster CMP‐sialic Acid Transporter

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Abstract
Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP‐sialic acid transporter (CMP‐Sia‐Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMPSia‐Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP‐Sia‐Tr. The expression of a hamster CMP‐Sia‐Tr fusion protein with an N‐terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP‐Sia‐Tr to the recently cloned human UDP‐galactose transporter (UDP‐Gal‐Tr). In contrast, sequence similarities to the yeast UDP‐N‐acetylglucosamine transporter (UDP‐GlcNAc‐Tr) and the GDP‐mannose transporter (GDP‐Man‐Tr) of Leishmania donovani are restricted to a region encoding the two most C‐terminally located transmembrane helices. A computer‐based structural analysis of CMPSia‐Tr proposes an eight transmembrane helix model with the N‐ and C‐termini located on the cytosolic side of the Golgi membrane.