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
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Date
1991
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Massey University
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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.
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Keywords
Lectins, Cyphomandra betacea, Tamarillo