Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans.
GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144-->Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144-->Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.
Strasser
Richard
R
Department für Angewandte Pflanzenwissenschaften und Pflanzenbiotechnologie, Institut für Angewandte Genetik und Zellbiologie, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Wien, Austria.
Stadlmann
Johannes
J
Svoboda
Barbara
B
Altmann
Friedrich
F
Glössl
Josef
J
Mach
Lukas
L
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D002460:Cell Line; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism*; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I