The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

Hans H. Wandall, Fernando Irazoqui, Mads Agervig Tarp, Eric P. Bennett, Ulla Mandel, Hideyuki Takeuchi, Kentaro Kato, Tatsuro Irimura, Ganesh Suryanarayanan, Michael A Hollingsworth, Henrik Clausen

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

Original languageEnglish (US)
Pages (from-to)374-387
Number of pages14
JournalGlycobiology
Volume17
Issue number4
DOIs
StatePublished - Apr 1 2007

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Glycosylation
Glycopeptides
Lectins
Carbohydrates
Peptides
Substrates
Substrate Specificity
Protein Isoforms
polypeptide N-acetylgalactosaminyltransferase
Ricin
Kinetics
Mucins
Enzymes
Polysaccharides

ASJC Scopus subject areas

  • Biochemistry

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The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc : Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. / Wandall, Hans H.; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P.; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik.

In: Glycobiology, Vol. 17, No. 4, 01.04.2007, p. 374-387.

Research output: Contribution to journalArticle

Wandall, Hans H. ; Irazoqui, Fernando ; Tarp, Mads Agervig ; Bennett, Eric P. ; Mandel, Ulla ; Takeuchi, Hideyuki ; Kato, Kentaro ; Irimura, Tatsuro ; Suryanarayanan, Ganesh ; Hollingsworth, Michael A ; Clausen, Henrik. / The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc : Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. In: Glycobiology. 2007 ; Vol. 17, No. 4. pp. 374-387.
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AU - Wandall, Hans H.

AU - Irazoqui, Fernando

AU - Tarp, Mads Agervig

AU - Bennett, Eric P.

AU - Mandel, Ulla

AU - Takeuchi, Hideyuki

AU - Kato, Kentaro

AU - Irimura, Tatsuro

AU - Suryanarayanan, Ganesh

AU - Hollingsworth, Michael A

AU - Clausen, Henrik

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N2 - Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

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