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.
Highlights
The hirudin-like factors (HLFs) represent an only recently described subclass of compounds derived from the salivary glands of medicinal leeches (Müller et al 2016, 2017)
We have described and functionally characterized several HLFs including HLF1-4 (Müller et al 2016, 2019, 2020a, b) and HLF5, HLF6, and HLF8
We constructed three different hybrids of the highly active thrombin inhibitor HLF1V and HLF6b: HLF-Hyb6a (N- and C-termini of HLF1V fused to the central globular domain of HLF6b), HLF-Hyb6b (N-terminus and central globular domain of HLF1V fused to the C-terminal tail of HLF6b), and HLF-Hyb6c (N- and C-termini of HLF6b fused to the central globular domain of HLF1V) (Fig. 2a, Table 1 and Table 2)
Summary
The hirudin-like factors (HLFs) represent an only recently described subclass of compounds derived from the salivary glands of medicinal leeches (Müller et al 2016, 2017). HLFs comprise structural (e.g., six cysteine residues within a central globular domain) and genetic (a gene structure composed of four exons and three introns) features that are characteristic for hirudins but may considerably differ from hirudins and among each other in biochemical properties like molecular weight (MW) or isoelectric point (pI value). Some HLF genes may encode different mRNAs (and different HLF molecules) due to alternative splice events. Neither the N-termini nor the central globular domains of the respective factors are altered, but the lengths and the amino acid composition of their C-terminal tails are different. In case of HLF4, the C-terminal tail of HLF4b conveyed thrombin-inhibitory potency to the molecule, whereas the tail of HLF4a did not (Müller et al 2020b). HLF6a, in contrast, has a very basic tail without any acidic amino acid residue
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