Abstract
A novel member of the mouse CMP-NeuAc: beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc V, was identified by BLAST analysis of expressed sequence tags. The sequence of the longest cDNA clone of ST6GalNAc V encoded a type II membrane protein with 8 amino acids comprising the cytoplasmic domain, 21 amino acids comprising the transmembrane region, and 306 amino acids comprising the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III and IV, with common amino acid sequences in sialyl motifs L and S among these three enzymes. Eleven CAG repeats were found in the stem region. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc V in a expression vector showed enzyme activity of alpha2,6-sialyltransferase almost exclusively for GM1b, but not toward glycoproteins. Sialidase treatment and thin layer chromatography immunostaining revealed that the product was GD1alpha. Northern blotting revealed that three transcripts of the gene were expressed specifically in brain tissues. It is concluded that this enzyme is involved in the synthesis of GD1alpha in the nervous tissues, and the CAG repeats may have implications in neurodegenerative diseases.
Highlights
A novel member of the mouse CMP-NeuAc: -N-acetylgalactosaminide ␣2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc V, was identified by BLAST analysis of expressed sequence tags
Isolation of ST6GalNAc V cDNA—Using the mouse expressed sequence tag data base, we found sequences
Since the ␣2,3 linkage-dominant sialidase from S. typhimurium LT2 has some activity toward ␣2,6-linked sialic acids, these results suggested that the structure of the sialylated intermediate product was NeuAc␣2, 6GalNAc1,4Gal1,4Glc-Cer, and the original product was GD1␣
Summary
Galactose; Sia, sialic acid; GalNAc, N-acetylgalactosamine; CMP-NeuAc, cytidine 5Ј-monophospho-N-. More than 15 species of sialyltransferase genes have been isolated (3, 4); and six genes for ␣2,3Gal (ST3Gal), one gene for ␣2,6Gal (ST6Gal), five genes for ␣2,8Sia (ST8Sia), and four genes for ␣2,6GalNAc (ST6GalNAc) have been cloned as sialyltransferase genes involved in the synthesis of sialylated carbohydrates on glycoproteins and glycolipids (4). ␣-Series gangliosides were defined as a new series of gangliosides containing NeuAc linked to the C6 position of GalNAc of the gangliotetraosyl backbone (6, 7) They have been thought only a minor component (8), and little is known about them. We have isolated a cDNA of GD1␣ synthase (ST6GalNAc V) gene expressed in the brain, which contains an interesting CAG repeat. ST6GalNAc V showed brain-specific expression, suggesting a critical role of ST6GalNAc V in the synthesis of GD1␣ in brain tissues
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