Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers

Abstract

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.

Isolation and Biochemical Characterization of Apios Tuber Lectin

“…The first 30 amino acid residues of band (c) containing ATL and Fraction A protein were determined to be AKLPFFSFNLDRFFPNEPNLIFQGDAKASS, which was completely consistent with the deduced N -terminal amino acid sequence of Apios americana lectin (AAL) [22]. Both ATL from SDS-PAGE and Fraction A protein separated by ion-exchange chromatography also showed the same N -terminal amino acid sequences, however, they gave slightly different peptide maps (Figure 5).…”

“…The peptide fragments were subjected to protein sequencing by a protein sequencer and MALDI-TOF-MS spectrometry. The amino acid sequence of ATL (the sequences of the fragments are not shown) was consisted with the deduced amino acid sequence of Apios tuber lectin (AAL) cDNA reported by Kouzuma et al [22], though the C -terminal amino acid sequence of ATL could not be determined in this study, due to the absence of the corresponding peptide fragments (Figure 6).…”

“…Asp (D) 240, Asp (D) 243, and Ser (S) 246 are putative truncating sites of SBA [24]. The amino acid sequence of AAL deduced from cDNA sequencing is cited from reference [22].…”

“…The ratio of the contents of Apios tube lectin (ATL) and non-lectin component (Fraction A protein) contained in the fractions was 1:4. Kouzuma et al [22] reported the purification and cDNA cloning of Apios tuber lectin, named AAL. AAL required the minimal concentration of 125 μg/mL to agglutinate rabbit erythrocytes and the hemagglutinating activity was not inhibited by any monosaccharide.…”

“…In fact, various bioactive molecules such as isoflavones have been reported in Apios tubers [17,18,19]. Furthermore, Zhang et al reported the purification, characterization and cDNA cloning of a Bowman-Birk type trypsin inhibitor [20,21] and a lectin [22] from Apios tubers.…”