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Mapping of a pepsin-resistant peptide and identification of glycosylation sites in the α subunit of the 11S globulin legumin from common bean.

Pajak A, Santamaria-Kisiel L, Marsolais F (2016) Mapping of a pepsin-resistant peptide and identification of glycosylation sites in the α subunit of the 11S globulin legumin from common bean. Oral presentation. Tenth Canadian Pulse Research Workshop, Winnipeg , October 25-28

Abstract

Unlike in soybean and other legumes, 11S globulins are relatively minor constituents of storage proteins in common bean (Phaseolus vulgaris). However, it was previously reported that the 11S globulin, legumin, contains a peptide of ca. 20 kDa which is resistant to simulated gastrointestinal digestion. In grain protein, resistance to proteolytic digestion often correlates with allergenicity. An approach combining purification and mass spectrometry was used for biochemical characterization. Using purified legumin, the peptide of ca. 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner and was mapped to an internal fragment of the α-subunit. The same fragment coincides with a peptide resistant to chymostrypsin digestion. The α-subunit of legumin exhibits a reduced mobility in SDS-PAGE, by a factor of ca. 10 kDa, and contains a consensus motif for plant O-glycosylation. Using a peanut agglutinin purified legumin, five contiguous sites of O-glycosylation were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) after trypsin digestion. Sites of O-glycosylation were identified as hydroxyproline residues substituted with one or two galactoses following fragmentation. For the site corresponding to the consensus motif, the mass of the O-glycosylated peptide was greater than the mass of the non-glycosylated version by ca. 3 kDa. In LC-MS/MS, this peptide co-eluted with a hexose chain of at least 10 residues. To our knowledge this represents the first detailed report of an O-glycosylated seed storage protein.

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