PRIMARY STRUCTURE OF THE 11.5 kDa LECTINE, BPL3, FROM THE MARINE GREEN ALGA BRYOPSIS PLUMOSA

Abstract

The N-acetyl-D-galactosamine and N-acetyl-D-glucosamine recognizing lectin (BPL3) from Bryopsis plumosa was purified by affinity chromatography. The recombinant protein was constructed based on cloned cDNA sequence. Purified native BPL3 had a molecular weight of 11.5 kDa on SDS-PAGE, also a molecular mass of 11533.85 Da by MALDI-TOF-MS. The hemagglutinating activity using human erythrocytes (types A, B, O) was inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. BPL3 consists of 106 amino acid residues including two cystein moieties and two N-glycosylation sites by prediction based on cDNA sequence. A primary structure was identified using MALDI-TOF-MS peptide mapping method in combination with specific peptidase (trypsin and carboxypeptidase Y), which was confirmed by the peptide sequences with MALDI-TOF-MS. In order to characterize the number and location of free Cys and disulfide bonds in the protein, chemical modification and MALDI-TOF-MS were used. C-terminal residues were determined by analyzing the molecular masses of the truncated peptides. The result show that BPL3 was composed of 103 amino acids and three amino acids in the C-terminal were cleaved. The peptide masses containing of N-glycosylation site were not changed in comparison with calculated peptide mass. This result suggested that the native BPL3 was not glycosylated. Our results also showed that presence of two cystein residues and two free sulfhydryls without disulfided bond in the amino acids sequence of BPL3. We will try to predict a complete primary structure of BPL3 through study of other types of post-translation modification.