Amplification and bioinformatics studies on iml-leb: lebestatin like peptide from iranian macrovipera lebetina snake

Shabnam Malekiha,1,* Hoda ayat,2 Ali mohammad ahadi,3

1. Shahrekord university
2. Shahrekord university
3. Shahrekord university

Abstract

Introduction

Angiogenesis, the development of new blood vessels, is a fundamental physiological process that promotes embryonic development, tissue repair and also promotes chronic inflammation, tumor growth and tumor metastasis. blood vessels facilitate tumor metastasis by serving as conduits for the transport of tumor cells to new sites. angiogenesis are regulated by integrins, which are members of a family of cell surface receptors and extracellular matrix proteins are their ligands. some integrins promote endothelial cell migration and survival during angiogenesis. several integrin-targeted therapeutic agents are currently in clinical trials for cancer therapy. snake venoms are natural sources of various biologically active compounds, with the major physiological role of killing and predigesting a prey. many viper venom proteins have been characterized as non-toxic, although displaying interesting biological properties. a number of snake venom proteins have the ability to interact with integrins. among these are the disintegrins, a family of small, non-enzymatic, and cysteine-rich proteins that found in the venom of numerous snake families. disintegrins can target specific integrins and therefore they could interfere in important processes involved in carcinogenesis, tumor growth, invasion and migration. lebestatin, a member of the lysine-threonine-serine (kts)-disintegrin family, was purified from macrovipera lebetina snake venom. it is a single-chain polypeptide composed of 41 amino acids. lebestatin interacts specifically with the α1β1integrin. it was thus able to inhibit both adhesion and migration of cancer cells. this disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect. so far, a few studies has been performed on component of iranian snake venom. the purpose of this study was to find and reproduce the coding gene of the lebestatin of the iranian species macrovipera lebetina.

Methods

Snake was collected by expert, the toxic glands was isolated and kept in -70 centigrade, until to extract rna. to design the primer, the homologue cdna sequence with lebestatin was derived from the national center for biotechnology information, then nucleotide and peptide blast was performed, and similar sequences were identified. depending to the nucleotide similarity, the forward and reverse primers were designed using gene runner software to amplify the gene. subsequently, the extraction of rna from the snakes gland was done. cdna was constructed by reverse transcriptase-pcr on rna with reverse transcriptase and oligo-dt primers. the pcr reaction was performed with forward primer complementary to signal peptide and reverse primer for the replication of lebestatin like gene. nested pcr was done by leading nesting primer based on mature peptide and reverse primer on reproductive sequence. after the reaction, 3 ml of the product to was investigated on 1% agarose gel and then was sequenced. bioinformatics studies were conducted to examine the second structure, using the pci-ss and i-tasser servers. the peptide was then modeled using modeller 9.19 software.

Results

In this study we report the sequence of the iranian macrovipera lebetina lebestatin gene for the first time from an iranian snake. the coding sequence of lebestatin gene include signal peptide, propeptide and mature peptide, involved 324 bp. the product of nested pcr contained mature peptide was 123 bp. the sequence analysis showed that iranian macrovipera lebetina lebestatin has a high sequence homology with known venom disintegrins such as lebestatin( macrovipera lebetina) obtustatin (vipera lebetina obtusa), viperistatin (vipera palestinae). the highest sequence identity was observed with kts motif-containing short disintegrins. indeed, iranian macrovipera lebetina lebestatin differs from lebestatin in only two amino acids flanking the kts loop and/or in the c-terminal part.

Conclusion

Disintegrins have numerous applications in studies on platelet thrombosis, endothelial cell apoptosis, migration and angiogenesis. here, we report the purification of a novel short disintegrin (41 amino acids), lebestatin, isolated from iranian macrovipera lebetina venom. the superimposition of the structural models of iranian macrovipera lebetina lebestatin with the 3d structure of lebestatin and obtustatin shows that they share similar conformational features. the main structural differences between the three peptides are located in the loop that contains the ktslts (iranian macrovipera lebetina lebestatin and obtustatin) or ktsrts (lebestatin) motif, and the c-terminal domain. (psfpa41-cooh, psypg41-cooh, and plypg41-cooh for iranian macrovipera lebetina lebestatin, lebestatin and obtustatin, respectively).

Keywords

Angiogenesis; integrins; disintegrins