Isolation and characterisation of a galactose-specific lectin from maturing seeds of lonchocarpus capassa and molecular cloning of the lectin gene

Abstract

A 29 kDa lectin that shows specificity for galactose was isolated from Lonchocarpus capassa seeds by a combination of ammonium sulphate precipitation and affinity chromatography on a galactose-sepharose column. The 29 kDa lectin subunit co-purified with a 45 kDa subunit. The N-terminal sequence of the 29 kDa subunit showed homology to other legume lectins while that of 45 kDa subunit was capped. A 360 bp fragment was amplified using degenerate primers designed from internal protein sequences of the 29 kDa subunit and a 5´ RACE system primer. The cDNA fragment was cloned into pTz57R/Tvector and transformed into E. coli. The partial amino acid sequence of the lectin subunit was deduced from the nucleotide sequence of the clone. The 360 bp fragment consisted of 342 bp sequence coding for the start codon, leader sequence, N-Terminal sequence and sequences of the 79 amino acids from N-terminus. Comparison of the deduced amino acid sequence with other legume lectins showed regions of sequence homology with precursor sequences of Robinia pseudoacacia Bark lectin, a non seed lectin from Pisum sativum (pea), and the galactose specific peanut agglutinin (PNA) from Arachis hypogaea. Alignment of these sequences showed conserved regions including the metal binding sites found in all legume lectins. The 5´ end DNA sequence was used to design locus-specific primers which were used with genome walking cassette primers in an attempt to amplify the full L. capassa lectin gene. The cassette primers were designed from restriction enzyme sites on the cassette. Of all the restriction enzymes on the cassette Hind III and the L. capassa gene-specific primers amplified 288 bp of the 342 bp sequence already obtained from sequencing of the cDNA sequence with minor amino acid differences. Although the full lectin sequence was not obtained the study confirmed the presence of a galactose-specific lectin in L. capassa seeds.