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.
Calvete
J J
JJ
Institut für Reproduktionsmedizin, Tierärztliche Hochschule Hannover, Hannover-Kirchrode, Germany. jcalvete@repro.tiho-hannover.de
Santos
C F
CF
Mann
K
K
Grangeiro
T B
TB
Nimtz
M
M
Urbanke
C
C
Sousa-Cavada
B
B
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D010450:Endopeptidases / Q000378:metabolism; D007887:Fabaceae / Q000737:chemistry*; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D037121:Plant Lectins; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D012639:Seeds / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D037102:Lectins; D009844:Oligosaccharides; D037121:Plant Lectins; D011134:Polysaccharides; D010450:Endopeptidases