PURIFICATION AND CHARACTERIZATION OF 3 ISOLECTINS OF SOYBEAN AGGLUTININ - EVIDENCE FOR C-TERMINAL TRUNCATION BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Soybean agglutinin (SBA) is a tetrameric D-Gal/D-GalAc-specific lectin possessing one Man, oligomannose-type chain/monomer. SBA exists as mu ltiple isolectins having similar binding and immunochemical properties . The present study shows that native SBA consists of at least five is olectins. Three of these isoforms have been purified by chromatofocusi ng and designated as SBA-I, SBA-II and SBA-III in order of their eluti on from a chromatofocusing column. The pI of the isolectins are 7.0, 6 .85 and 6.7, respectively, as determined by isoelectric focusing. Each isolectin was denatured in 6 M guanidine hydrochloride into their ind ividual subunits which were separated by reverse-phase high performanc e liquid chromatography (RP-HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) al ong with a minor peak (peak 2). The first peak of SBA-II existed as a doublet labeled as 1a and 1b. Each peak was analyzed by electrospray i onization mass spectrometry to characterize each isoform and determine their structural differences. The calculated mass of an intact lectin monomer from the amino acid sequence (253 residues) derived from cDNA of the lectin including a Man, oligomannose chain is 29438Da. The pre sent results show that peak 3 of each isoform corresponds to an intact subunit (alpha) while peak 1 of each isoform shows lower masses which are assigned to C-terminal fragmentation of the protein. Peak 1 of SB A-I has a molecular mass of 28000Da corresponding to a fragmented subu nit (beta) consisting of 240 residues (calculated molecular mass 28001 Da). Peak 1a of SBA-II shows a molecular mass of 28000Da corresponding to a fragmented beta subunit, while peak 1b showed two major species: a 28000-Da (beta subunit) and a 28327-Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a gamma subunit. In addition, peak 1b showed the presence of a molecular species of 28 627Da corresponding to a 246-residue subunit (gamma'). Peak 1 of SBA-I II showed a major molecular species corresponding to a fragmented gamm a subunit. The minor peak in the HPLC profile (peak 2) represented a s ubunit of 252 residues for all three isoforms. The results suggest tha t the subunit compositions of SPA-I, SBA-Il and SBA-m are approximatel y alpha(2) beta(2), alpha(2) beta gamma and alpha(2) gamma(2), respect ively. The results account for the decreasing pI of SBA-I to SBA-III s ince the 240-residue beta subunit involves loss of two acidic amino ac ids (Asp243 and Glu251) while the 243-residue gamma subunit involves l oss of one acidic amino acid (Glu251) from the intact subunit. The pre sent findings thus demonstrate that three isolectins of SBA arise from different combinations of intact and C-terminal fragmented subunits.