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Hematophagous leeches express a broad variety of secretory factors in their salivary glands; among them are hirudins, inhibitors of blood coagulation, and decorsins/ornatins, inhibitors of platelet aggregation. Here, we describe the identification and molecular and functional characterization of putative hirudins and decorsins/ornatins in two leech species of American origin, Limnobdella mexicana and Haementeria vizottoi. The leech species represent two orders of leeches, the proboscis-bearing Rhynchobdellida and the non-proboscis-bearing Arhynchobdellida. Members of the hirudin superfamily, such as hirudins or decorsins/ornatins, are described for the first time in the genus Haementeria. Both species expressed very potent inhibitors of platelet aggregation, but only the putative hirudins of L. mexicana displayed high thrombin-inhibitory potency, whereas the putative hirudin of H. vizottoi turned out to be a hirudin-like factor. The results of our study provide new insights into the evolutionary background of the blood-sucking lifestyle in leeches.
The aquatic gastropod Theodoxus fluviatilis occurs in Europe and adjacent areas of Asia. The snail species has formed two genetically closely related subgroups, the freshwater ecotype (FW) and the brackish water ecotype (BW). Other than individuals of the FW ecotype, those of the BW ecotype survive in salinities of up to 28‰. Coastal aquatic ecosystems may be affected by climate change due to salinization. Thus, we investigated how the two Theodoxus ecotypes adjust to changes in environmental salinity, focusing on the question whether Na+/K+-ATPase or V-ATPase are regulated on the transcriptional, the translational or at the activity level under changing external salinities. Animals were gradually adjusted to extreme salinities in containers under long-day conditions and constant temperature. Whole body RNA- or protein extracts were prepared. Semi-quantitative PCR- and western blot-analyses did not reveal major changes in transcript or protein abundances for the two transporters under low or high salinity conditions. No significant changes in ATPase activities in whole body extracts of animals adjusted to high or low salinity conditions were detected. We conclude that constitutive expression of ATPases is sufficient to support osmotic and ion regulation in this species under changing salinities given the high level of tolerance with respect to changes in body fluid volume.
Abstract
The neritid snail Theodoxus fluviatilis has formed regional subgroups in northern Europe, where it appears in both freshwater (FW) and brackish water (BW) in coastal areas of the Baltic Sea. These ecotypes show clear differences in osmotolerance and in the modes of accumulating organic osmolytes under hyperosmotic stress. We reasoned that the expression patterns of soluble proteins in the two ecotypes may differ as well. BW snails have to deal with a higher salinity (up to 20‰) than FW snails (0.5‰) and also cope with frequent fluctuations in environmental salinity that occur after heavy rains or evaporation caused by extended periods of intense sunshine. Therefore, the protein expression patterns of specimens collected at five different FW and BW sites were analyzed using 2D SDS‐PAGE, mass spectrometry, and sequence comparisons based on a transcriptome database for Theodoxus fluviatilis. We identified 89 differentially expressed proteins. The differences in the expression between FW and BW snails may be due to phenotypic plasticity, but may also be determined by local genetic adaptations. Among the differentially expressed proteins, 19 proteins seem to be of special interest as they may be involved in mediating the higher tolerance of BW animals towards environmental change compared with FW animals.
Abstract
Background
Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non‐hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system.
Objective
Whether the thrombin inhibitor hirudin, probably the most prominent leech‐derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study.
Methods
We identified several putative hirudin‐encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin‐inhibitory potencies in coagulation assays.
Results
We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin‐like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM.
Conclusions
Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non‐hematophagous leech.
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.