Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500046, India.
Institute for Hygiene, Westfälische Wilhelms-University, Münster, D-48149, Germany.
Int J Biol Macromol. 2014 Aug;69:400-7. doi: 10.1016/j.ijbiomac.2014.05.073. Epub 2014 Jun 4.
An affinity purified mannose/glucose specific lectin from the seeds of Dolichos lablab (Indian bean/lablab bean) resolves into five subunits upon SDS-PAGE in the range of Mr 12-20kDa. Partial de novo sequencing of subunits resulted in 88% and 73% sequence coverage for α and β subunits of the cDNA derived FRIL (Flt3 receptor interacting lectin) sequence, respectively and suggested that four bands correspond to the α-subunits while the band of lowest molecular mass is designated as β. It was proposed in an earlier study on FRIL that the difference in molecular mass of α-subunits is due to differences in C-terminal processing and differential N-glycosylation i.e. numbers of N-glycans present (Colucci et al., 1999). Thus, differential N-glycosylation of the purified mannose/glucose specific lectin was unravelled by in-gel trypsin/chymotrypsin digestion of the α-subunits followed by desalting and ZIC-HILIC enrichment of N-glycopeptides. Subsequently, analyses by nano electrospray ionisation quadrupole time of flight mass spectrometry and low-energy collision-induced dissociation experiments revealed the presence of a typical paucimannose type N-glycan (Man2(Xyl)GlcNAc2(Fuc)) in α subunits 2-4.
从菜豆（印度豆/菜豆）种子中纯化得到的甘露糖/葡萄糖特异性凝集素在 SDS-PAGE 中解析为 Mr 12-20kDa 范围内的五个亚基。亚基的从头部分测序导致 cDNA 衍生的 FRIL（Flt3 受体相互作用凝集素）序列的α和β亚基的序列覆盖率分别为 88%和 73%，并表明四个条带对应于α-亚基，而分子量最低的条带被指定为β。在 FRIL 的早期研究中提出，α-亚基的分子量差异是由于 C 末端加工和差异 N-糖基化的差异，即存在的 N-聚糖数量不同（Colucci 等人，1999 年）。因此，通过α-亚基的胶内胰蛋白酶/糜蛋白酶消化、脱盐和 ZIC-HILIC 富集 N-糖肽，揭示了纯化的甘露糖/葡萄糖特异性凝集素的差异 N-糖基化。随后，通过纳升电喷雾离子化四极杆飞行时间质谱和低能量碰撞诱导解离实验分析，揭示了α亚基 2-4 中存在典型的寡甘露糖型 N-聚糖（Man2(Xyl)GlcNAc2(Fuc))。
