Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

TY - JOUR

T1 - Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

AU - Zlocowski, Natacha

AU - Sendra, Victor G

AU - Lorenz, Virginia

AU - Villarreal, Marcos A

AU - Jorge, Alberto

AU - Núñez, Yolanda

AU - Bennett, Eric P

AU - Clausen, Henrik

AU - Nores, Gustavo A

AU - Irazoqui, Fernando J

N1 - Copyright © 2011 Elsevier Inc. All rights reserved.

PY - 2011/6/24

Y1 - 2011/6/24

N2 - Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (" ricin-like" ) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.

AB - Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (" ricin-like" ) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.

KW - Acetylation

KW - Amino Acid Sequence

KW - Catalysis

KW - Humans

KW - Molecular Sequence Data

KW - N-Acetylgalactosaminyltransferases

KW - Polysaccharides

KW - Protein Binding

KW - Protein Conformation

U2 - 10.1016/j.bbrc.2011.05.125

DO - 10.1016/j.bbrc.2011.05.125

M3 - Journal article

C2 - 21651894

SN - 0006-291X

VL - 410

SP - 140

EP - 145

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 1

ER -