Molecular cloning and characterization of ConBr, the lectin of Canavalia brasiliensis seeds.
ConBr, a lectin isolated from Canavalia brasiliensis seeds, shares with other legume plant lectins from the genus Canavalia (Diocleinae subtribe) primary carbohydrate recognition specificity for D-mannose and D-glucose. However, ConBr exerts different biological effects than concanavalin A, the lectin of Canavalia ensiformis seeds, regarding induction of rat paw edema, peritoneal macrophage spreading in mouse, and in vitro human lymphocyte stimulation. The primary structure of ConBr was established by cDNA cloning, amino acid sequencing, and mass spectrometry. The 237-amino-acid sequence of ConBr displays Ser/Thr heterogeneity at position 96, indicating the existence of two isoforms. The mature Canavalia brasiliensis lectin monomer consists of a mixture of predominantly full-length polypeptide (alpha-chain) and a small proportion of fragments 1-118 (beta-chain) and 119-237 (gamma-chain). Although ConBr isolectins and concanavalin A differ only in residues at positions 58, 70, and 96, ConBr monomers associate into dimers and tetramers in a different pH-dependent manner than those of concanavalin A. The occurrence of glycine at position 58 does not allow formation of the hydrogen bond that in the concanavalin A tetramer exists between Asp58 of subunit A and Ser62 of subunit C. The consequence is that the alpha carbons of the corresponding residues in ConBr are 1.5 A closer that in concanavalin A, and ConBr adopts a more open quaternary structure than concanavalin A. Our data support the hypothesis that substitution of amino acids located at the subunit interface of structurally related lectins of the same protein family can lead to different quaternary conformations that may account for their different biological activities.
Grangeiro
T B
TB
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Brazil.
Schriefer
A
A
Calvete
J J
JJ
Raida
M
M
Urbanke
C
C
Barral-Netto
M
M
Cavada
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004487:Edema / Q000209:etiology; D007887:Fabaceae / Q000235:genetics*; D017343:Genes, Plant; D005998:Glycine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D037121:Plant Lectins; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D012639:Seeds
D017931:DNA Primers; D037102:Lectins; D037121:Plant Lectins; D005998:Glycine