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Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I

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Abstract

Isolation and purification of a α-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 ∼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 ~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 × 10−6 M) and BA (1.09 × 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.

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Abbreviations

PBS:

phosphate-buffered saline

nSL-I/SL-I:

native peanut stem lectin

re-SL-I:

recombinant SL-I

Con A:

Concanavalin A

LBL:

lima bean lectin

WBA I and WBA II:

winged bean basic- and acidic- agglutinin respectively

PHA-E:

Phaseolus vulgaris erythroagglutinin

PAL:

Pterocarpus angolensis man/glc specific seed lectin

SBA:

soybean lectin

ANS:

1,8-anilino-1-naphthalenesulfonic acid

BA:

6-benzylaminopurine

PMSF:

phenylmethylsulfonylfluoride

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Acknowledgements

Authors gratefully acknowledge Dr. Souvik Maiti for his guidance in fluorescence studies. Authors also acknowledge the Director of IGIB for providing facilities to carry out this work.

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Authors and Affiliations

  1. Unit of Proteomics and Comparative Genomics, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi, 110 007, India

    Monika Pathak, Bharat Singh, Amit Sharma, Praveen Agrawal, Santosh B. Pasha, Hasi R. Das & Rakha H. Das

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  1. Monika Pathak
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  2. Bharat Singh
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  3. Amit Sharma
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  4. Praveen Agrawal
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  5. Santosh B. Pasha
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  6. Hasi R. Das
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  7. Rakha H. Das
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Corresponding author

Correspondence to Rakha H. Das.

Additional information

The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under the Accession No. AJ585523

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Pathak, M., Singh, B., Sharma, A. et al. Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I. Plant Mol Biol 62, 529–545 (2006). https://doi.org/10.1007/s11103-006-9038-6

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