Abstract
A novel member of the mouse CMP-NeuAc:beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of alpha2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1alpha, GQ1balpha, and GT1aalpha. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of alpha-series gangliosides in the nervous tissues and many other tissues.
Highlights
Sialic acid-containing glycosphingolipids are designated gangliosides and have been thought to play important roles in a wide variety of biological events such as cell-cell or cell-extracellular matrix interaction, protein targeting, and acceptance of extracellular molecules and particles [1]
Isolation of ST6GalNAc VI cDNA—To clone GT1a␣ and GQ1b␣ synthase, the National Center for Biotechnology Information (NCBI) data bank of EST cDNA clones was probed with the deduced amino acid sequence of the mouse ST6GalNAc IV
The sequence was similar to but distinct from that of the previously cloned sialyltransferases, suggesting that the clone encoded a novel member of the sialyltransferase gene family
Summary
Galactose; Sia, sialic acid; GalNAc, N-acetylgalactosamine; CMP-NeuAc, cytidine 5Ј-monophospho-Nacetylneuraminic acid; mAb, monoclonal antibody; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified Eagle’s minimum essential medium; BSM, bovine submaxillary mucin. More than 16 species of sialyltransferase genes utilizing glycoproteins and/or glycolipids as an acceptor have been cloned [2], i.e. 6 genes for ␣2,3Gal (ST3Gal), 1 gene for ␣2,6Gal (ST6Gal), 5 genes for ␣2,8Sia (ST8Sia), and 5 genes for ␣2,6GalNAc (ST6GalNAc) Many of these sialyltransferase genes were isolated by polymerase chain reaction based on similar sequences named sialyl motifs, which were first identified by Paulson and co-workers [3] in purified sialyltransferases, and in all sialyltransferases isolated thereafter [2]. We have isolated a novel member of the ST6GalNAc gene subfamily designated ST6GalNAc VI This enzyme is specific for glycolipid acceptors and can synthesize all ␣-series gangliosides so far defined. The expression pattern of the gene is much broader than that of ST6GalNAc V and is distinct from that of other members of the ST6GalNAc subfamily
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