A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana.
In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.
Strasser
Richard
R
Institute of Applied Genetics and Cell Biology, BOKU, University of Natural Resources and Applied Life Sciences, A-1190 Viena, Austria. richard.strasser@boku.ac.at
Bondili
Jayakumar Singh
JS
Vavra
Ulrike
U
Schoberer
Jennifer
J
Svoboda
Barbara
B
Glössl
Josef
J
Léonard
Renaud
R
Stadlmann
Johannes
J
Altmann
Friedrich
F
Steinkellner
Herta
H
Mach
Lukas
L
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D004269:DNA, Bacterial; D000939:Epitopes / Q000737:chemistry; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006031:Glycosylation; D007313:Insecta; D007983:Lewis Blood Group Antigens; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009844:Oligosaccharides / Q000737:chemistry*; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D021381:Protein Transport; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D000939:Epitopes; D007983:Lewis Blood Group Antigens; C494219:Lewis a oligosaccharide; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; C036483:T-DNA; D005700:Galactosyltransferases; C524433:Galt1 protein, Arabidopsis