Characterizing the link between glycosylation state and enzymatic activity of the endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana.
Characterizing the link between glycosylation state and enzymatic activity of the endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana.
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DOI：
10.1074/jbc.m113.475558
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发表时间：
2013-08-02
期刊：
影响因子：
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通讯作者：
Strasser R
中科院分区：
文献类型：
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作者：
Liebminger E;Grass J;Altmann F;Mach L;Strasser R
背景：N-糖基化影响内切-β-1，4-葡聚糖酶KORRIGAN1的生物合成，KORRIGAN1是植物细胞壁合成的关键。结果：低糖基化KORRIGAN1的酶活性降低。当N-葡聚糖组成发生变化时，不会观察到这一点。结论：改良N-葡聚糖加工对足底KORRIGAN1功能无直接影响。意义：揭示了几个N-糖基化位点与KORRIGAN1活性的相关性。N-糖基化和N-糖链加工的缺陷经常导致植物细胞壁结构的变化，包括纤维素的结构变化，纤维素是植物中含量最丰富的多糖。KORRIGAN1(KOR1)是拟南芥中一种在纤维素生物合成过程中起重要作用的糖蛋白酶。KOR1是一种膜锚定的内切β1，4-葡聚糖酶，它的胞外区含有8个潜在的N-糖基化位点。在这里，我们在昆虫细胞中表达了一种可溶性的、具有酶活性的蛋白，并分析了它的N-糖基化状态。结构分析表明，所有8个潜在的N-糖基化位点都被利用了。单独消除进化上保守的N-糖基化位点并不会取消正确的KOR1折叠，但ASN-216、ASN-324、ASN-345和ASN-567的突变会导致酶活性显著降低。相反，在I类α-甘露糖苷酶抑制剂Kifunensine的存在下生产野生型KOR1，取消了KOR1N-糖链到复杂结构的转换，并不影响酶的活性。为了在更自然的条件下研究KOR1的N-糖基化位点占有率和N-糖链组成，我们在烟草叶片中表达了KOR1-Fc-GFP融合蛋白。虽然ASN-108和ASN-133携带低聚糖苷N-连接的寡糖，但其他6个糖基化位点被复杂的N-糖链修饰。有趣的是，由A.thaliana rsw2-1等位基因编码的部分功能KOR1 G429R突变体在N.benthamiana中表达时只显示出寡糖结构，表明它保留在内质网中。综上所述，我们的数据表明，几个N-糖基化位点的利用对KOR1的活性很重要，而连接的N-糖链的结构并不是关键。
Background: N-Glycosylation affects the biosynthesis of the endo-β1,4-glucanase KORRIGAN1 which is crucial for cell wall synthesis in plants. Results: Underglycosylated KORRIGAN1 displays reduced enzymatic activity. This is not observed upon changes to the N-glycan composition. Conclusion: Modified N-glycan processing exerts no direct effect on KORRIGAN1 function in planta. Significance: The relevance of several N-glycosylation sites for KORRIGAN1 activity is revealed. Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.