Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I.
Isolation and purification of a alpha-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282-285]. Native SL-I is a glycoprotein having approximately 31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests' amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a approximately 30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced "delayed leaf senescence" and "cotyledon expansion". Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 x 10(-6 )M) and BA (1.09 x 10(-5 )M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.
Pathak
Monika
M
Unit of Proteomics and Comparative Genomics, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India.
Singh
Bharat
B
Sharma
Amit
A
Agrawal
Praveen
P
Pasha
Santosh B
SB
Das
Hasi R
HR
Das
Rakha H
RH
D000595:Amino Acid Sequence; D010367:Arachis / Q000737:chemistry*; D001483:Base Sequence; D001593:Benzyl Compounds; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003583:Cytokinins / Q000037:antagonists & inhibitors*; D017931:DNA Primers; D018076:DNA, Complementary; D007701:Kinetin / Q000494:pharmacology*; D037102:Lectins / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D011687:Purines; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001593:Benzyl Compounds; D003583:Cytokinins; D017931:DNA Primers; D018076:DNA, Complementary; D037102:Lectins; D011687:Purines; C480551:benzylaminopurine; D007701:Kinetin