Purification and characterization of a lectin from tamarillo fruits (Cyphomandra betacea) : a thesis presented in partial fulfilment of requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

Abstract

Lectins specific in their binding to oligomers of [beta]1,4 linked N-acetylglucosamine were identified in the fruits of Cyphomandra species of the family Solanaceae. Thus, Cyphomandra species can be considered as a new source of lectins for basic and applied studies.

New lectins (designated as CBL1 and CBL2 ) were identified from tamarillo fruits (Cyphomandra betacea). CBL1 was purified. Biochemical characterization, subcellular localization and molecular sequence analysis for this new lectin were made. CBL2, which was immunologically unrelated to CBL1, was not further characterized.

CBL1 could be readily purified using affinity and ion exchange chromatography. CBL1 comprised two subunits joined by non convalant interactions. Subunit size was 25 kDa. N,N,'N",N""-tetraacetylchitotetraose was the most effective carbohydrate for inhibition of CBL1 induced agglutination of rabbit erythrocytes. CBL1 consists of abundant residues of Cys (16%), Gly (14%), Glx (13%), Ser (11%), Pro (9%) and Asx (7%), and to a lesser extent, hydroxyproline residues.

CBL1 was found to be an abundant, extremely stable and developmentally regulated protein. It was found predominantly in cell walls of fruit tissues using immunofluorescence techniques. CBL1 could play a defence role in seed development.

Despite the general resemblance of chemical composition and carbohydrate specificities, no cross-reaction among solanaceous lectins in double immunodiffusion tests performed in gels containing their carbohydrate ligands was demonstrated, suggesting they may not have similar epitopes.

Four tryptic peptides and the N-terminal fragment of CBL1 were sequenced, which showed some homologies with the Gramineaelectins. Since CBL1 and the Graminea electins shared similar properties such as amino acid composition and sugar specificities, it is suggested that CBL1, a solanaceous lectin, might be evolutionarily related to the Gramineae lectins.

Two cDNA clones were isolated with anti-CBL1 serum, and sequenced. One of them (X200), which reacted weakly with anti-CBL1 serum, was 96% identical with a bacterial gene ilvC encoding acetohydroxy acid isomeroreductase. The peptide encoded by this cDNA could have some similar epitopes to CBL1, which resulted in its isolation. Another clone (X208), which showed stronger reaction with anti-CBL1 serum, was found to contain putative peptide sequences which did not show homology with CBL1 peptide sequences. This clone could be derived from one domain of CBL1's coding region, while the peptide sequences could be confined to another domain. Complexity in immunoscreening the clone encoding CBL1 is discussed, and future work on the isolation of cDNA clone encoding this interesting lectin is suggested.