Strasser Richard, Stadlmann Johannes, Svoboda Barbara, Altmann Friedrich, Glössl Josef, Mach Lukas
Department für Angewandte Pflanzenwissenschaften und Pflanzenbiotechnologie, Institut für Angewandte Genetik und Zellbiologie, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Wien, Austria.
Biochem J. 2005 Apr 15;387(Pt 2):385-91. doi: 10.1042/BJ20041686.
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.
N-乙酰葡糖胺基转移酶I（GnTI）是一种驻留在高尔基体中的酶，对于高甘露糖型聚糖加工成杂合型和复合型N-聚糖至关重要。拟南芥cgl突变体缺乏GnTI活性，因此积累了寡甘露糖苷结构。对cgl GnTI cDNA进行分子克隆发现了一个点突变，该突变导致关键氨基酸替换（Asp144→Asn），从而产生了一个额外的N-糖基化位点。在昆虫细胞中对cgl GnTI进行异源表达证实了其缺乏活性以及该N-糖基化位点的使用情况。值得注意的是，将Asp144→Asn突变引入兔GnTI（该突变不会导致形成新的N-糖基化位点）后，产生了一种酶活性大幅降低但仍可检测到的蛋白质。在cgl植物中表达Asn144兔GnTI可以部分恢复复合型N-聚糖的形成。这些结果表明，cgl植物中GnTI活性的完全缺乏主要是由于额外的N-聚糖，它似乎干扰了该酶的正确折叠。
