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Cloning and characterization of mannose binding jacalin related lectin encoding gene from Indian Cycas (Cycas annaikalensis Singh and Radha)

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Abstract

Mannose specific jacalin-related lectins or agglutinins (mJRLs) constitute an important superfamily of proteins known to play vital roles in various biological processes. In the present study, a cDNA having 876 bp open reading frame (ORF) coding for mJRL of 291 amino acids residues was cloned from pinna of Cycas annaikalensis which is endemic to Western Ghats, India and designated as C. annaikalensis pinna lectin (CAPL). Expression of the coding sequence under the control of a T7 promoter in E. coli produced 31 kDa protein. The purified recombinant protein had shown agglutination with erythrocytes of rabbit blood. The deduced amino acid sequence of CAPL showed two sugar binding sites (also determined to be jacalin-like lectin domains) and 95% similarity with C. revoluta leaf lectin (CRLL) protein. Further, a monomeric protein of CAPL consisting of mannose binding residues and jacalin motifs that are having 35–90% similarities with mJRLs which have already been reported. A phylogenetic tree exhibited the grouping of CAPL into a subclade different from that of the CRLL. Also, a model of cycas leaf lectin was built by homology modeling using 1ZGRA (Parkia platycephala seed lectin) as a template for the construction of three-dimensional structures. Structural modeling and docking studies were completed using Discovery studio version 2.1. This study, first of its kind, reports mJRLs from the Indian gymnosperm.

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Abbreviations

mJRLs:

Mannose binding jacalin related lectins

CAPL:

Cycas annaikalensis pinna lectin

CRLL:

Cycas revoluta leaf lectin

gJRLs:

Galactose binding JRLs

SOPMA:

Self-optimized prediction method in alignment

RMSD:

Root mean square deviation

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Acknowledgements

Author Dr. P. Radha thanks University Grant Commission (UGC), Govt. of India for financial support through UGC-PDF for women scheme (PDFWM-2011-12-GE-TAM-9600(SA-II). Prof. V. D. Reddy and Prof. K.V. Rao, Centre for Plant Molecular Biology (CPMB), Osmania University, Hyderabad and Dr. M. Shesheer Kumar, VSS Pavan Kumar and M. Ravi Chintada, RAS life sciences Pvt Ltd, Hyderabad, India are acknowledged for their constant support and suggestion during this study.

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Authors and Affiliations

  1. Centre for Plant Molecular Biology, Osmania University, Hyderabad, Telangana, 500007, India

    P. Radha & Rajasekhar Urla

  2. Siddha Clinical Research Unit, Palayamkottai, Tamil Nadu, 627002, India

    P. Radha

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  1. P. Radha
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  2. Rajasekhar Urla
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Contributions

RU designed and conducted the gene isolation; cloning and biochemical characterization studies. PR created the whole idea, experiments and performed cloning, bioinformatics analysis. RU, PR drafted the manuscript. Both the authors read and approved the final manuscript.

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Correspondence to P. Radha.

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Supplementary Fig. 1

Amplification of C. annaikalensis pinnae lectin gene (CAPL): M. Gene Ruler 1 kbDNA ladder; 1, 2 CAPL amplified product at ~ 876 bp (TIFF 40927 kb)

Supplementary Fig. 2

Restriction digestion analysis of pTNOT-CAPL: M. GeneRuler 1 kb DNA ladder; 1, 2, and 3. release of ~ 900 bp band corresponding to CAPL gene and vector backbone at 3 kbp up on digestion with Bam HI and Not I (TIFF 36268 kb)

Supplementary Fig. 3

Restriction analysis of pET28a+ CAPL: M. GeneRuler DNA ladder mix; 1. Undigested pET28a+ CAPL plasmid; 2. 2, 3, 4 release of ~ 900 bp band corresponding to CAPL gene up on digestion with Bam HI and Not I (TIFF 33317 kb)

Supplementary Fig. 4

The full-length cDNA sequence and deduced amino acid sequence of CAPL. The start codon (ATG) is in italics and the stop codon (TGA) is in italics and underlined. The underlined sequence is the sugar binding sites; the bold residues indicate the potential glycosylation sites; the shades residues are GXXXD motifs (TIFF 31839 kb)

Supplementary Fig. 5

Secondary structure analysis of CAPL using SOPMA secondary structure prediction method: CAPL 291 amino acid contribution in the form of α helices (54 aa), beta turns (27 aa), random coil (124 aa) and extended strand (86 aa) were represented by blue, purple, green and red lines respectively (TIFF 5108 kb)

Supplementary Fig. 6

Phylogenetic analysis of CAPL with other mJRLs: Heltuba-Helianthus tuberosus lectin, Calsepa-Calystegia sepium lectin;CCA-Castanea crenata; MornigaM-Morus nigra; PK-Parkia platycephala lectin; Banlec-Musa acuminate; KM + -Artocarpus integrifolia lectin; PAL-Phlebodium aureum lectin; Orysata-Oryza sativa lectin; CRLL-Cycas revoluta leaf lectin; C-a. CAPL; C.c.-CCPL; C-ru-C. rumphii lectin (TIFF 3511 kb)

Supplementary Fig. 7

Ramachandran plot analysis of modelled structure of CAPL protein showed 90.1% residues are favored regions, whereas 7.2% and 2.7% of the protein in allowed regions and in the outlier region respectively (TIFF 26621 kb)

Supplementary Fig. 8

Three dimensional structure of CAPL with their sugar binding residues (amino acids 139-143 and 277-281) (TIFF 32791 kb)

Supplementary material 9 (DOCX 15 kb)

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Radha, P., Urla, R. Cloning and characterization of mannose binding jacalin related lectin encoding gene from Indian Cycas (Cycas annaikalensis Singh and Radha). J. Plant Biochem. Biotechnol. 29, 314–322 (2020). https://doi.org/10.1007/s13562-019-00513-3

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