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 Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalpha1-3)GlcNAc, a typical plant Nglycan. 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 with 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.

Comparison of the amino acid sequences of the lectins from seeds of Dioclea lehmanni and Canavalia maritima.

The amino acid sequences of the major lectins from the seeds of Dioclea lehmanni and Canavalia maritima were determined by DABITC/PITC microsequence analysis of peptides derived from the proteins by enzymatic digestions with trypsin, chymotrypsin and the protease from S. aureus V8. These sequences were found to be very similar to those of the lectins from Dioclea grandiflora and Canavalia ensiformis (Con A). The D. lehmanni lectin was unusual amongst legume lectins in that it contained a single Cys.

The amino acid sequences of the alpha 1 and alpha 2 subunits of the isolectins from seeds of Lathyrus ochrus (L) DC.

The complete amino acid sequences of the alpha 1 and alpha 2 subunits of the isolectins (LoL I and LoL II) from seeds of Lathyrus ochrus were determined by analysis of peptides derived from the proteins by digestion with trypsin, chymotrypsin and the S. aureus V8 protease. Both subunits consisted of single polypeptide chains of 53 amino acids, which differed from one another in only 4 positions near their C-termini, and exhibited high homology to the light (alpha) chains of the lectins from Lathyrus sativus, L. odoratus and a number of other legume seeds.