Galactosyltransferases from Arabidopsis thaliana in the biosynthesis of type II arabinogalactan: molecular interaction enhances enzyme activity.
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.
Adiphol
A
Dilokpimol
Christian Peter
CP
Poulsen
György
G
Vereb
Satoshi
S
Kaneko
Alexander
A
Schulz
Department of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871 Frederiksberg, C, Denmark. nge@plen.ku.dk.
Naomi
N
Geshi
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005685:Galactans / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000378:metabolism
D029681:Arabidopsis Proteins; D005685:Galactans; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C044347:1,6-galactan; C000603836:GALT29A protein, Arabidopsis; D005700:Galactosyltransferases; C005653:arabinogalactan; D005690:Galactose