Amino acid sequence, glycan structure, and proteolytic processing of the lectin of Vatairea macrocarpa seeds

VML is a galactose-binding lectin isolated from Vatairea macrocarpa seeds. By SDS-polyacrylamide gel electrophoresis, VML is a glycoprotein composed of a major 32-34 kDa double band (alpha-chain) and minor 22 kDa and 13 kDa bands. N-terminal sequencing of electroblotted samples showed that the 22 and 13 kDa bands corresponded to C-(beta) and N-(gamma) terminal fragments of the alpha-chain, respectively. The primary structure of VML displays similarity with other leguminous lectins, particularly with Erythrina variegata, Robinia pseudoacacia and Sophora japonica lectins. VML is N-glycosylated at asparagine residues at positions 111 and 183 with one major glycan structure. Tandem mass spectrometry and methylation analysis indicated the presence of Man alpha 1-6[(Man alpha 1-3)(Xyl beta 1-2)]Man beta 1-4-GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc, a typical plant N-glycan. Equilibrium sedimentation analysis by analytical centrifugation showed that VML had a mass of 122-130 kDa, which did not change within the pH range 2.5-8.5. These data indicated that VML is a pH-independent homotetrameric protein and that a small proportion of the alpha-subunits is cleaved into noncovalently associated N- and C-terminal fragments. Mass spectrometric analysis suggested a mechanism for the proteolytic processing of VML. V. macrocarpa lectin contains a mixture of doubly (28 525 Da) and singly (27 354 Da) glycosylated alpha-chains. Deglycosylation of Asn-111 correlates,vith proteolytic cleavage of the Asn-114-Lys-115 bond yielding glycosylated gamma (residues 1-114, 12 304 Da) and nonglycosylated beta-(residues 115-239, 14 957 Da) chains. Some beta-chain molecules are further deglycosylated and N-terminally processed yielding products of molecular masses of 13 783 Da and 13 670 Da. (C) 1998 Federation of European Biochemical Societies.