Short tail stories: the hirudin-like factors HLF6 and HLF7 of the Asian medicinal leech, Hirudinaria manillensis

Abstract

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

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

Fig. 1

Multiple sequence alignment of hirudin variants HV1 of Hirudo medicinalis and HM3 of Hirudinaria manillensis and HLF variants HLF1V, HLF5, HLF8, HLF6, HLF6d, HLF7a, and HLF7b. The alignments were generated using the CLS Sequence Viewer software package v8.0 (CLC bio, Aarhus, Denmark). Black background indicates conserved residues; gray background indicates similar residues. The six conserved cysteine residues giving rise to the three dimensional structure are marked in bold, acidic amino acid residues are marked in red, and basic amino acid residues are marked in blue. The PKP and DFxxIP motifs are boxed. Abbreviations are used according to the IUPAC code

Fig. 2

a Schematic representation of the N-termini, the central globular domains, and the C-terminal tails of HLF1V (light blue), HLF6a (light red), HLF6b (purple), HLF3s (dark blue), the hybrid variants HLF-Hyb6a-c, and the C-terminal truncated variants HLF1V-DDK and HLF-Hyb3s. b Schematic representation of the N-termini, the central globular domains, and the C-terminal tails of HLF1V (light blue), HLF7a (light green), and HLF7b (red) and the hybrid variants HLF-Hyb7a-e. The yellow color indicates an alternative N-terminus of HLF7-Hyb7c and HLF-Hyb7e derived from the nucleotide sequence of the HLF7 gene (see text for details)

Fig. 3

Standard blood coagulation assays using the thrombin time assay (TT) of HLF6d as well as the hybrids HLF-Hyb6a, HLF-Hyb6b, and HLF-Hyb6c. n = 3–5, error bars indicate SD. *: p < 0.05

Fig. 4

Standard blood coagulation assays using the thrombin time assay (TT) of HLF variants HLF7a and HLF7b as well as the hybrids HLF-Hyb7a, HLF-Hyb7b, and HLF-Hyb7c. n = 3–5, error bars indicate SD. *: p < 0.05; **: p < 0.01; ***: p < 0.001

Fig. 5

Standard blood coagulation assays using the thrombin time assay (TT) of HLF1V, HLF1V-DDK, and HLF-Hyb3s. n = 3–5, error bars indicate SD. *: p < 0.05; **: p < 0.01; ***: p < 0.001