Purification and characterization of three isolectins of soybean agglutinin. Evidence for C-terminal truncation by electrospray ionization mass spectrometry.
Soybean agglutinin (SBA) is a tetrameric D-Gal/D-GalNAc-specific lectin possessing one Man9 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 double 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 Man9 oligomannose chain is 29438 Da. The present 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 SBA-I has a molecular mass of 28000Da corresponding to a fragmented subunit (beta) consisting of 240 residues (calculated molecular mass 28001Da). 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 28627Da corresponding to a 246-residue subunit (gamma'). Peak 1 of SBA-III showed a major molecular species corresponding to a fragmented gamma 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 alpha 2 beta 2, alpha 2 beta gamma and alpha 2 gamma 2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Mandal
D K
DK
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Nieves
E
E
Bhattacharyya
L
L
Orr
G A
GA
Roboz
J
J
Yu
Q T
QT
Brewer
C F
CF
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019791:Guanidine; D006146:Guanidines; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037121:Plant Lectins; D011489:Protein Denaturation; D030262:Soybean Proteins; D010835:Phytohemagglutinins
D019791:Guanidine; D006146:Guanidines; D037102:Lectins; D046911:Macromolecular Substances; D037121:Plant Lectins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins