Purification and characterization of three isolectins of soybean agglutinin

Soybean agglutinin (SBA) is a tetrameric d D‐Gal/ d ‐GalNAc‐specific lectin possessing one Man 9 oligomannose‐type chain/monomer. SBA exists as multiple isolectins having similar binding and immunochemical properties. The present study shows that native SBA consists of at least five isolectins. Three of these isoforms have been purified by chromatofocusing and designated as SBA‐I, SBA‐II and SBA‐III in order of their elution 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 individual subunits which were separated by reverse‐phase high performance liquid chromatography (RP‐HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) along with a minor peak (peak 2). The first peak of SBA‐II existed as a doublet labeled as 1 a and 1 b. Each peak was analyzed by electrospray ionization 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 9 oligomannose chain is 29438 Da. The present results show that peak 3 of each isoform corresponds to an intact subunit (α) while peak 1 of each isoform shows lower masses which are assigned to C‐terminal fragmentation of the protein. Peak 1 of SBA‐I has a molecular mass of 28000Da corresponding to a fragmented subunit (β) consisting of 240 residues (calculated molecular mass 28001Da). Peak 1 a of SBA‐II shows a molecular mass of 28000Da corresponding to a fragmented β subunit, while peak 1 b showed two major species: a 28000‐Da (β subunit) and a 28327‐Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a γ subunit. In addition, peak 1 b showed the presence of a molecular species of 28627Da corresponding to a 246‐residue subunit (γ′). Peak 1 of SBA‐III showed a major molecular species corresponding to a fragmented γ subunit. The minor peak in the HPLC profile (peak 2) represented a subunit of 252 residues for all three isoforms. The results suggest that the subunit compositions of SBA‐I, SBA‐II and SBA‐III are approximately α 2 β 2 , α 2 βγ and α 2 γ 2 , respectively. The results account for the decreasing pI of SBA‐I to SBA‐III since the 240‐residue β subunit involves loss of two acidic amino acids (Asp243 and Glu251) while the 243‐residue γ subunit involves loss of one acidic amino acid (Glu251) from the intact subunit. The present findings thus demonstrate that three isolectins of SBA arise from different combinations of intact and C‐terminal fragmented subunits.