Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis

Abstract

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Keywords: Blood coagulation; Hirudin; Hirudin-like factors; Hirudinaria manillensis; Medicinal leeches; Tandem-Hirudin.

Fig. 1

Multiple sequence alignment of hirudin-variant 1 (HV1) and hirudin-like factor HLF1V from Hirudo medicinalis, hirudin-variant HM1 and HLF5 from Hirudinaria (Poecilobdella) manillensis and domain-I (TH1) and domain-II (TH2) of Tandem-Hirudins TH and PmTH (a putative TH encoded by Poecilobdella manillensis). Black background indicates conserved residues; grey background indicates similar residues. Acidic amino acid residues are labelled in red, basic amino acid residues in blue. A red arrow indicates the signal peptide cleavage site. The six conserved cysteine residues giving rise to the three dimensional structure are marked in bold and labelled in yellow. Boxes indicate the structural and functional domains of hirudin HV1: signal peptide (grey), N-terminus (green), central globular domain (yellow) and elongated C-terminal tail (red). Abbreviations are used according to the IUPAC code

Fig. 2

Schematic representation of HV1 (upper part) and TH (lower part). Acidic amino acid residues are labelled in red, basic amino acid residues in blue and cysteine residues in yellow. Putative disulfide bonds are indicated by bold lines

Fig. 3

Functional characterisation of bacterially synthesized r-TH and hirudin variant 1 (r-HV1) in blood coagulation (A), trypsin activity (B) and platelet aggregation (C) assays. Measurements for blood coagulation were cancelled after 300 s and considered as a complete inhibition of coagulation. Concentrations used in the assays were 3.2 µmol/l (A), 0.4 μmol/l (B) and 3.8 μmol/l (C), respectively. aPTT indicates the activated partial thromboplastin time test, PT the prothrombin time test and TT the thrombin time test

Fig. 4

Standard blood coagulation assays using the thrombin time test (TT) of cell-free synthesized TH, hirudin HV1 and HLF5 with ( +) or without ( −) the addition of disulfide bond enhancer during the synthesis reaction in bacterial cell lysates. Lysates containing either an empty vector or a vector containing the coding region of the dihydrofolate reductase (DHFR) served as the negative controls. Aliquots of synthesis reactions used for the measurements were either 5 µl (n = 1, right) of 10 µl (1 µl for HV1) (n = 3, left)

Fig. 5

Schematic representation of a segment on genome contig00065 of Hirudinaria (Poecilobdella) manillensis comprising four putative hirudin/HLF genes (Hiru_V1–V4) and one putative tandem hirudin gene (PmTH). Red arrows indicate position, size and orientation of the respective genes. The upper boxes illustrate the structures of two genes (left: putative hirudin/HLF Hiru_V1; right: putative tandem hirudin PmTH). Exons (E) are labelled in green, and introns (I) are labelled in red. The bar indicates a length of 1 kb

Fig. 6

Schematic drawing visualizing the multiplication hypothesis. TH indicates the tandem hirudin, OH a putative molecule with multiple hirudin-like domains. Exon-overlapping codons are illustrated by an angled connection