Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.
The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.
von Schaewen
A
A
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Sturm
A
A
O'Neill
J
J
Chrispeels
M J
MJ
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003433:Crosses, Genetic; D005643:Fucose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis*
D006023:Glycoproteins; D011134:Polysaccharides; D005643:Fucose; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D005944:Glucosamine