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

Natacha Zlocowski; Victor G Sendra; Virginia Lorenz; Marcos A Villarreal; Alberto Jorge; Yolanda Núñez; Eric P Bennett; Henrik Clausen; Gustavo A Nores; Fernando J Irazoqui

doi:10.1016/j.bbrc.2011.05.125

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

Natacha Zlocowski, Victor G Sendra, Virginia Lorenz, Marcos A Villarreal, Alberto Jorge, Yolanda Núñez, Eric P Bennett, Henrik Clausen, Gustavo A Nores, Fernando J Irazoqui

4 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Biochemical and Biophysical Research Communications
Volume	410
Issue number	1
Pages (from-to)	140-5
Number of pages	6
ISSN	0006-291X
DOIs	https://doi.org/10.1016/j.bbrc.2011.05.125
Publication status	Published - 24 Jun 2011

Keywords

Acetylation
Amino Acid Sequence
Catalysis
Humans
Molecular Sequence Data
N-Acetylgalactosaminyltransferases
Polysaccharides
Protein Binding
Protein Conformation

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10.1016/j.bbrc.2011.05.125

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title = "Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation",

abstract = "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.",

keywords = "Acetylation, Amino Acid Sequence, Catalysis, Humans, Molecular Sequence Data, N-Acetylgalactosaminyltransferases, Polysaccharides, Protein Binding, Protein Conformation",

author = "Natacha Zlocowski and Sendra, {Victor G} and Virginia Lorenz and Villarreal, {Marcos A} and Alberto Jorge and Yolanda N{\'u}{\~n}ez and Bennett, {Eric P} and Henrik Clausen and Nores, {Gustavo A} and Irazoqui, {Fernando J}",

year = "2011",

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doi = "10.1016/j.bbrc.2011.05.125",

language = "English",

volume = "410",

pages = "140--5",

journal = "Biochemical and Biophysical Research Communications",

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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

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 -

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

Abstract

Keywords

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