Galactosyltransferases from arabidopsis thaliana in the biosynthesis of type II arabinogalactan: Molecular interaction enhances enzyme activity

Adiphol Dilokpimol, Christian P. Poulsen, G. Vereb, Satoshi Kaneko, Alexander Schulz, Naomi Geshi

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Background: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.Results: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses β-1,6-galactosyltransferase activity, elongating β-1,6-galactan side chains and forming 6-Gal branches on the β-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Förster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of β-1,6-galactosyltransferase activities compared to AtGALT29A alone.Conclusions: AtGALT29A is a β-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to β-1,6-galactan and to β-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.

Original languageEnglish
Article number90
JournalBMC Plant Biology
Volume14
Issue number1
DOIs
Publication statusPublished - Apr 3 2014

Fingerprint

galactosyltransferases
Galactosyltransferases
arabinogalactans
arabinogalactan proteins
Arabidopsis
Arabidopsis thaliana
galactans
biosynthesis
enzyme activity
galactose
Enzymes
Galactose
Galactans
Polysaccharides
polysaccharides
Nicotiana benthamiana
glycosylation
enzymes
Glycosylation
Uridine Diphosphate Galactose

Keywords

  • Arabidopsis thaliana
  • Arabinogalactan protein
  • FRET
  • Galactosyltransferase
  • Golgi apparatus
  • Plant cell wall
  • Protein O-glycosylation
  • Protein-protein interaction

ASJC Scopus subject areas

  • Plant Science
  • Medicine(all)

Cite this

Galactosyltransferases from arabidopsis thaliana in the biosynthesis of type II arabinogalactan : Molecular interaction enhances enzyme activity. / Dilokpimol, Adiphol; Poulsen, Christian P.; Vereb, G.; Kaneko, Satoshi; Schulz, Alexander; Geshi, Naomi.

In: BMC Plant Biology, Vol. 14, No. 1, 90, 03.04.2014.

Research output: Contribution to journalArticle

Dilokpimol, Adiphol ; Poulsen, Christian P. ; Vereb, G. ; Kaneko, Satoshi ; Schulz, Alexander ; Geshi, Naomi. / Galactosyltransferases from arabidopsis thaliana in the biosynthesis of type II arabinogalactan : Molecular interaction enhances enzyme activity. In: BMC Plant Biology. 2014 ; Vol. 14, No. 1.
@article{9b63dad78a7e473d9068f69c9ab08b9c,
title = "Galactosyltransferases from arabidopsis thaliana in the biosynthesis of type II arabinogalactan: Molecular interaction enhances enzyme activity",
abstract = "Background: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.Results: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses β-1,6-galactosyltransferase activity, elongating β-1,6-galactan side chains and forming 6-Gal branches on the β-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by F{\"o}rster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of β-1,6-galactosyltransferase activities compared to AtGALT29A alone.Conclusions: AtGALT29A is a β-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to β-1,6-galactan and to β-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.",
keywords = "Arabidopsis thaliana, Arabinogalactan protein, FRET, Galactosyltransferase, Golgi apparatus, Plant cell wall, Protein O-glycosylation, Protein-protein interaction",
author = "Adiphol Dilokpimol and Poulsen, {Christian P.} and G. Vereb and Satoshi Kaneko and Alexander Schulz and Naomi Geshi",
year = "2014",
month = "4",
day = "3",
doi = "10.1186/1471-2229-14-90",
language = "English",
volume = "14",
journal = "BMC Plant Biology",
issn = "1471-2229",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Galactosyltransferases from arabidopsis thaliana in the biosynthesis of type II arabinogalactan

T2 - Molecular interaction enhances enzyme activity

AU - Dilokpimol, Adiphol

AU - Poulsen, Christian P.

AU - Vereb, G.

AU - Kaneko, Satoshi

AU - Schulz, Alexander

AU - Geshi, Naomi

PY - 2014/4/3

Y1 - 2014/4/3

N2 - Background: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.Results: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses β-1,6-galactosyltransferase activity, elongating β-1,6-galactan side chains and forming 6-Gal branches on the β-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Förster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of β-1,6-galactosyltransferase activities compared to AtGALT29A alone.Conclusions: AtGALT29A is a β-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to β-1,6-galactan and to β-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.

AB - Background: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.Results: We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses β-1,6-galactosyltransferase activity, elongating β-1,6-galactan side chains and forming 6-Gal branches on the β-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Förster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of β-1,6-galactosyltransferase activities compared to AtGALT29A alone.Conclusions: AtGALT29A is a β-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to β-1,6-galactan and to β-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.

KW - Arabidopsis thaliana

KW - Arabinogalactan protein

KW - FRET

KW - Galactosyltransferase

KW - Golgi apparatus

KW - Plant cell wall

KW - Protein O-glycosylation

KW - Protein-protein interaction

UR - http://www.scopus.com/inward/record.url?scp=84898024932&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84898024932&partnerID=8YFLogxK

U2 - 10.1186/1471-2229-14-90

DO - 10.1186/1471-2229-14-90

M3 - Article

C2 - 24693939

AN - SCOPUS:84898024932

VL - 14

JO - BMC Plant Biology

JF - BMC Plant Biology

SN - 1471-2229

IS - 1

M1 - 90

ER -