title: Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.
abstract: Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.
authors.affiliation: Department of Biochemistry, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Souichi
authors.initials: S
authors.lastname: Koyota
authors.forename: Yu
authors.initials: Y
authors.lastname: Myoen
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Naoki
authors.initials: N
authors.lastname: Yatabe
authors.forename: Yukio
authors.initials: Y
authors.lastname: Koizumi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Aosasa
authors.forename: Norihisa
authors.initials: N
authors.lastname: Nishimichi
authors.forename: Haruo
authors.initials: H
authors.lastname: Matsuda
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Sugiyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000276:immunology*; D019151:Peptide Library; D010455:Peptides / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D019151:Peptide Library; D010455:Peptides; D057127:Single-Chain Antibodies; D017350:N-Acetylgalactosaminyltransferases