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

FEBS Letters

Mann, Karlheinz¹  Urbanke, Claus³  Calvete, Juan J⁵  Sousa-Cavada, Benildo⁴  Grangeiro, Thalles B⁴  Nimtz, Manfred²  Santos, Cláudia F⁴ 

[1] Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany;Gesellschaft für biotechnologische Forschung (GBF) mbH, Mascheroder Weg 1, 38124 Braunschweig, Germany;Biophysikalisch-biochemische Verfahren, Medizinische Hochschule, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany;Departamento de Bioquı́mica e Biologia Molecular, Universidade Federal do Ceará, C.P. 6020, 60451-970 Fortaleza, Brazil;Institut für Reproduktionsmedizin, Tierärztliche Hochschule Hannover, Bünteweg 15, 30559 Hannover-Kirchrode, Germany

关键词: Vatairea macrocarpa seed;    Galactose-binding leguminous lectin;    Primary structure;    Carbohydrate structure;    Mass spectrometry;    Posttranslational processing;   

DOI  :  10.1016/S0014-5793(98)00243-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Manα1-6[(Manα1-3)(Xylβ1-2)]Manβ1-4-GlcNAcβ1-4(Fucα1-3)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 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.

【 授权许可】

Unknown