Elsevier

Biochimie

Volume 112, May 2015, Pages 49-56
Biochimie

Research paper
Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis

https://doi.org/10.1016/j.biochi.2015.02.011Get rights and content

Highlights

  • A novel RGD peptide identified in Lutzomyia ayacuchensis saliva was characterized.

  • The RGD peptide inhibited platelet aggregations in the RGD sequence-dependent manner.

  • Additionally, it inhibited blood coagulation pathway irrespective of the RGD sequence.

  • The RGD peptide is considered to play an important role in the blood feeding.

Abstract

Sequence analysis of the Lutzomyia (Lu.) ayacuchensis salivary gland cDNA library identified a short peptide containing an RGD (Arg-Gly-Asp) sequence flanked by two cysteine residues in the C-terminal end as the most abundant transcript. In the present study, a recombinant protein of the RGD-containing peptide, designated ayadualin, was expressed in Escherichia coli and its activity was characterized. Ayadualin inhibited both collagen and ADP-induced platelet aggregations by interfering with the binding of integrin αIIbβ3 to fibrinogen. The RGD sequence and cysteine residues located on both sides of the RGD sequence were essential for the inhibitory action. Moreover, ayadualin efficiently inhibited the intrinsic blood coagulation pathway irrespective of the RGD sequence. Measuring the enzymatic activity of coagulation factors using chromogenic substrates revealed that ayadualin efficiently inhibited factor XIIa (FXIIa) activity in a dose-dependent manner. In addition, pre-incubation of ayadualin with FXII inhibited FXIIa activity, while activated FXIIa was not affected by ayadualin, indicating that ayadualin inhibits the activation of FXII, but not enzymatic activity of FXIIa. These results indicated that ayadualin plays an important role in the blood feeding of Lu. ayacuchensis by inhibiting host hemostasis via dual mechanisms.

Introduction

Hematophagous insects have evolved a wide set of pharmacologically active molecules to counteract host hemostatic processes [1], [2], [3]. When probing in the host skin before blood feeding, they inject saliva, a cocktail of bioactive agents, containing anticoagulants, vasodilators, and inhibitors of platelet aggregation [1], [2], [3], [4], [5], [6]. Other salivary molecules almost certainly involved in the feeding process include anti-inflammatory and immunosuppressive molecules [1], [2], [3], [4], [5], [6]. Since hematophagous arthropods have evolved their feeding strategies independently, each species has developed unique pharmacologically active agents in their saliva to overcome host hemostatic defenses [1], [2], [3], [4], [5], [6].

Phlebotomine sand flies are hematophagous insects of the family Psychodidae in the order Diptera, and some of them transmit Leishmania protozoa, the causative agent of leishmaniasis [7], [8]. In addition to the antihemostatic activity, sand fly saliva exacerbates the infection of Leishmania parasites in mammalian hosts [9], [10], [11]. To date, the profiles of salivary components have been defined in 6 Old World Phlebotomus species; Phlebotomus papatasi, Phlebotomus ariasi, Phlebotomus perniciosus, Phlebotomus argentipes, Phlebotomus duboscqi, Phlebotomus arabicus, and Phlebotomus orientalis [12], [13], [14], [15], [16], [17] and 3 Lutzomyia species; Lutzomyia (Lu.) longipalpis, Lutzomyia ayacuchensis, and Lutzomyia intermedia [18], [19], [20], [21]. In the salivary transcriptome analysis of Lu. ayacuchensis, a proven vector of Leishmania (Leishmania) mexicana in Ecuadorian Andes [22], [23], [24] and Leishmania (Viannia) peruviana in Peruvian Andes [24], [25], a peptide containing an RGD (Arg-Gly-Asp) sequence flanked by cysteine residues in the C-terminal end was identified as the most abundant transcript. A homologous protein has been identified in the salivary gland transcriptome of Lu. longipalpis (LuloRGD); however, the function remains to be characterized [18].

The RGD sequence present in adhesive proteins is recognized by several integrins, and binding of fibrinogen to integrin αIIbβ3 (glycoprotein GPIIb-IIIa) expressed on activated platelets via the RGD sequences is a crucial mechanism for platelet aggregation [26]. Thus, molecules containing RGD sequences have the potential to inhibit platelet aggregation by interfering with the binding of platelets to fibrinogen. The natural RGD-containing peptide, disintegrin, is a family of cysteine-rich peptides containing RGD motifs, discovered originally in snake venoms and later in saliva of hematophagous animals such as leeches and ticks [27], [28], [29]. Disintegrins present their RGD sequences to integrins by forming a characteristic disulfide bond-stabilized loop, the formation of which is essential for their activity, and competitively interfere with the binding between fibrinogen and integrins resulting in inhibition of platelet aggregation [27], [28], [29]. In insects, this family of proteins has been found solely in the salivary glands of the horsefly Tabanus yao [29], [30]. On the other hand, a short RGD-containing peptide was identified from Lu. ayacuchensis and it has an RGD sequence in the C-terminal end, which differs from disintegrin family proteins. Additionally, it has only two cysteine residues located on both sides of the C-terminal RGD sequence, which is uncommon in disintegrins. In the present study, a recombinant protein of the short RGD-containing peptide from Lu. ayacuchensis salivary glands, designated ayadualin, was prepared, and its biological activity was characterized.

Section snippets

Sequence analysis

The sequences were aligned with CLUSTAL W software [31] and examined using the program MEGA (Molecular Evolutionary Genetics Analysis) version 5.1 [32]. A phylogenetic tree by the neighbor-joining method was constructed with the distance algorithms available in the MEGA package. Bootstrap values were determined on 1000 replicates of the data sets.

Production of recombinant proteins

A DNA fragment encoding full length of mature ayadualin was amplified and inserted into the EcoRI site of N-terminal thioredoxin (Trx)-hexahistidine

Sequence analysis of the RGD-containing peptide, ayadualin

A short peptide containing an RGD (Arg-Gly-Asp) sequence flanked by cysteine residues in the C-terminal end was identified as the most abundant transcript in the Lu. ayacuchensis salivary glands [20]. This peptide, named ayadualin, (GenBank accession number: AK416785) coded for 67 amino acids containing a 20 amino acid signal peptide with a predicted molecular mass of 5.3 kDa in the mature form (Fig. 1A). The homologous proteins identified so far are salivary RGD-containing peptides from Lu.

Discussion

Ayadualin was identified as the most abundant transcript in salivary glands of Lu. ayacuchensis [20]. The peptide shared homology with salivary RGD peptides from Lu. longipalpis (LuloRGD) and Lu. intermedia (Linb-1 and Linb-2) [18], [19], [21], but not with other proteins, suggesting that these peptides are unique to Lutzomyia species. Although these peptides are expected to function as platelet aggregation inhibitors via their RGD sequences, their functions have not been characterized. In this

Conclusion

In the present study, functional characterization of a recombinant salivary RGD peptide from Lu. ayacuchensis, named ayadualin, revealed that ayadualin affects host hemostasis by dual mechanisms; inhibition of platelet aggregation and an anticoagulant action in the contact phase. Therefore, this peptide is considered to play an important role in the blood feeding process of Lu. ayacuchensis. Because of its unique structure, further structural analysis of the peptide may help understanding of

Conflict of interest

The authors have declared that there is no conflict of interest.

Author contributions

H.K. designed the study, performed experiments, and drafted the manuscript; E.A.G. contributed to the sample preparation; M.F., Y.I. and H.I. performed experiments; H.U. and T.M. analyzed the data and contributed to the statistics; Y.H. edited the manuscript.

Acknowledgments

This study was supported in part by Grants-in-aid for Scientific Research from Japan Science and Technology Agency (JST), A-STEP feasibility study program (No. AS242Z00081Q), and the Ministry of Education, Science, Culture and Sports of Japan (No. 23580424).

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