Expression cloning of mouse cDNA of CMP-NeuAc:lactosylceramide α2,3- sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Satoshi Fukumoto, Hiroshi Miyazaki, George Goto, Takeshi Urano, Keiko Furukawa, Koichi Furukawa

Research output: Contribution to journalArticlepeer-review

62 Citations (Scopus)

Abstract

Expression cloning of a cDNA for the α2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of α2,3- sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among α2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

Original languageEnglish
Pages (from-to)9271-9276
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number14
DOIs
Publication statusPublished - Apr 2 1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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