Gnanesh Kumar B S, Pohlentz G, Mormann M, Siva Kumar N
Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500046, India.
Protein Expr Purif. 2013 May;89(1):7-15. doi: 10.1016/j.pep.2013.02.004. Epub 2013 Feb 16.
α-Mannosidase is a key enzyme in processing and degradation of N-glycans in plants and animals. In the present study α-mannosidase from crude extracts of Dolichos lablab (Indian beans) has been purified by ammonium sulfate precipitation, anion exchange, galactose Sepharose, phenyl Sepharose, gel permeation and Con A Sepharose chromatography. The purified protein migrated as a single band corresponding to 116 kDa on SDS-PAGE under reducing conditions. The pH and temperature optima of α-mannosidase activity determined by use of p-nitrophenyl-α-D-mannopyranoside as substrate were found to be 5.0 and 60-65°C, respectively. The KM was 1.48 mM and swainsonine was a potent inhibitor of the enzyme with IC(50) value 50-80 nM. Additionally, the de novo amino acid sequencing showed active site regions highly conserved among other plant acidic α-mannosidases and yielded sequence coverage of approximately 32.5%. N-glycopeptide analysis revealed the presence of paucimannosidic type structure in a conserved N-glycosylation site as well as at least one oligo mannosidic glycan at an undetermined site after ZIC-HILIC enrichment of proteolytic glycopeptides. The partial biochemical and molecular characterization of this enzyme reveals that it is a class II α-mannosidase from the glycosyl hydrolase family 38.
α-甘露糖苷酶是动植物中N-聚糖加工和降解的关键酶。在本研究中，通过硫酸铵沉淀、阴离子交换、半乳糖琼脂糖、苯基琼脂糖、凝胶渗透和伴刀豆球蛋白A琼脂糖层析，从白扁豆（印度豆）粗提物中纯化了α-甘露糖苷酶。在还原条件下，纯化后的蛋白质在SDS-PAGE上迁移为一条对应于116 kDa的单带。以对硝基苯基-α-D-甘露吡喃糖苷为底物测定的α-甘露糖苷酶活性的最适pH和温度分别为5.0和60 - 65°C。米氏常数为1.48 mM，苦马豆素是该酶的有效抑制剂，IC(50)值为50 - 80 nM。此外，从头氨基酸测序显示活性位点区域在其他植物酸性α-甘露糖苷酶中高度保守，序列覆盖率约为32.5%。N-糖肽分析表明，在保守的N-糖基化位点存在寡甘露糖型结构，并且在蛋白酶解糖肽经ZIC-HILIC富集后的一个未确定位点存在至少一种寡聚甘露糖型聚糖。该酶的部分生化和分子特征表明它是糖基水解酶家族38中的II类α-甘露糖苷酶。
