Makoto Kiso

Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

MOLECULAR CLONING OF BRAIN-SPECIFIC GD1ALPHA SYNTHASE (ST6GALNAC V) CONTAINING CAG/GLUTAMINE REPEATS

A novel member of the mouse CMP-NeuAc: β-N-acetylgalactosaminide α2,6-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 α2,6-sialyltransferase almost exclusively for GM1b, but not toward glycoproteins. Sialidase treatment and thin layer chromatography immunostaining revealed that the product was GD1α. 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 GD1α in the nervous tissues, and the CAG repeats may have implications in neurodegenerative diseases.

Molecular cloning and expression of mouse GD1α/GT1aα/GQ1bα synthase (ST6GalNAc VI) gene

A novel member of the mouse CMP-NeuAc:β-N-acetylgalactosaminide α2,6-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 α2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1α, GQ1bα, and GT1aα. 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 α-series gangliosides in the nervous tissues and many other tissues.

Chapter 8:Carbohydrate-derived Organoselenium Compounds: Synthesis and Application in the Structural Analysis of Biomolecules

This chapter deals with the synthesis of the carbohydrate-derived organoselenium compounds and their utility in the structural analysis of biomolecules. The isotopic distribution and anomalous scattering properties of selenium encouraged chemists towards selenium chemistry and its incorporation into organic molecules. Selenium is ideal for anomalous scattering in multi-wavelength anomalous dispersion and single-wavelength anomalous dispersion of X-ray crystallographic analysis of biomolecules and proteins. Furthermore, the 77Se isotope is nuclear magnetic resonance (NMR)-active with the spin quantum number ½. Experimental resolution of the atomic structure of the proteins is advantageous, to understand molecular mechanisms as well as to fuel further research in drug development. In this regard, X-ray crystal analysis of selenium-incorporated proteins plays a vital role in the structural elucidation. Over two-thirds of the protein structures have been elucidated using this technique. This chapter summarizes the synthesis of carbohydrate-derived organoselenium compounds and the site-specific selenium derivatization of selenocarbohydrates, selenonucleosides and their chemical applications. X-ray crystallographic analysis and 77Se NMR spectroscopy are two powerful tools with which to analyse the 3-D structures of biomolecules in detail.