Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.)

The 11S globulin legumin typically accounts for approximately
3%
of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately
20 kDa is resistant to pepsin digestion. Sequence prediction suggested
that the pepsin-resistant peptide is located at the C-terminal end
of the α-subunit, within a glutamic acid-rich domain, overlapping
with a chymotrypsin-resistant peptide. Using purified legumin, the
peptide of approximately 20 kDa was found to be resistant to pepsin
digestion in a pH-dependent manner, and its location was determined
by two-dimensional gel electrophoresis and LC-MS-MS. The location
of the chymotrypsin-resistant peptide was confirmed by immunoblotting
with peptide-specific polyclonal antibodies. The presence of a consensus
site for proline hydroxylation and arabinosylation, the detection
of hydroxyproline residues, purification by lectin affinity chromatography,
and a difference in electrophoretic migration between the chymotrypsin-
and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

11S球蛋白腿肌蛋白通常占普通豆类（Phaseolus vulgaris）总蛋白的约3%。已有研究表明，一种约20 kDa的腿肌蛋白肽对胃蛋白酶消化具有抵抗力。序列预测表明，该胃蛋白酶抵抗肽位于α亚基的C端，在一个富含谷氨酸的区域内，与一种胰凝乳蛋白酶抵抗肽部分重叠。利用纯化的腿肌蛋白，发现该约20 kDa的肽在pH依赖性方式下对胃蛋白酶消化具有抵抗力，其位置通过二维凝胶电泳和LC-MS-MS确定。通过使用针对肽特异性多克隆抗体的免疫印迹技术，证实了胰凝乳蛋白酶抵抗肽的位置。存在脯氨酸羟基化和阿拉伯糖基化的一致位点、羟基脯氨酸残基的检测、通过凝集素亲和层析纯化，以及胰凝乳蛋白酶和胃蛋白酶抵抗肽在电泳迁移率上的差异，均提示这些肽内存在一个大的O-糖基化。