The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.
- MeSH
- antigeny CD18 MeSH
- bakteriální adheze MeSH
- bakteriální adheziny metabolismus MeSH
- Bordetella pertussis * metabolismus MeSH
- faktory virulence rodu Bordetella MeSH
- glykosaminoglykany MeSH
- hemaglutininy metabolismus MeSH
- heparin MeSH
- integriny MeSH
- lidé MeSH
- makrofágový antigen 1 MeSH
- pertuse * MeSH
- proteasy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Fibrinogen-related proteins with lectin activity are believed to be part of the tick innate immune system. Several fibrinogen-related proteins have been described and characterised mainly on the basis of their cDNA sequences while direct biochemical evidence is missing. One of them, the haemolymph lectin Dorin M from the tick Ornithodoros moubata was isolated and characterised in more depth. RESULTS: Several fibrinogen-related proteins were detected in the haemolymph of ixodid ticks Dermacentor marginatus, Rhipicephalus appendiculatus, R. pulchellus, and R. sanguineus. These proteins were recognised by sera directed against the tick lectin Dorin M and the haemagglutination activity of the ticks R. appendiculatus and D. marginatus. Cross-reactivity of the identified proteins with antibodies against the fibrinogen domain of the human ficolin was also shown. The carbohydrate-binding ability of tick haemolymph was confirmed by haemagglutination activity assays, and this activity was shown to be inhibited by neuraminic acid and sialylated glycoproteins as well as by N-acetylated hexosamines. The fibrinogen-related proteins were shown to be glycosylated and they were localised in salivary glands, midguts, and haemocytes of D. marginatus. Hemelipoglycoprotein was also recognised by sera directed against the fibrinogen-related proteins in all three Rhipicephalus species as well as in D. marginatus. However, this protein does not contain the fibrinogen domain and thus, the binding possibly results from the structure similarity between hemelipoglycoprotein and the fibrinogen domain. CONCLUSIONS: The presence of fibrinogen-related proteins was shown in the haemolymph of four tick species in high abundance. Reactivity of antibodies directed against ficolin or fibrinogen-related proteins with proteins which do not contain the fibrinogen domain points out the importance of sequence analysis of the identified proteins in further studies. Previously observed expression of fibrinogen-related proteins in haemocytes together with the results of this study suggest involvement of fibrinogen-related proteins in tick immunity processes. Thus, they have potential as targets for anti-tick vaccines and as antimicrobial proteins in pharmacology. Research on fibrinogen-related proteins could reveal further details of tick innate immunity processes.
- MeSH
- anatomické struktury zvířat chemie MeSH
- Dermacentor chemie MeSH
- fibrinogen imunologie MeSH
- glykoproteiny imunologie metabolismus MeSH
- hemaglutininy imunologie metabolismus MeSH
- hmyzí proteiny imunologie metabolismus MeSH
- lektiny imunologie metabolismus MeSH
- lidé MeSH
- metabolismus sacharidů MeSH
- Ornithodoros chemie MeSH
- protilátky imunologie MeSH
- Rhipicephalus chemie MeSH
- vazba proteinů MeSH
- zkřížené reakce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Tick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa). The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity. RESULTS: Hemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc(2)Hex(9), HexNAc(2)Hex(10), HexNAc(4)Hex(7), and HexNAc(4)Hex(8). The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata. It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms. CONCLUSION: A 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.
- MeSH
- Dermacentor chemie metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- glykoproteiny chemie izolace a purifikace metabolismus MeSH
- glykosylace MeSH
- hemaglutininy chemie izolace a purifikace metabolismus MeSH
- hemoproteiny chemie izolace a purifikace metabolismus MeSH
- imunoprecipitace MeSH
- lipoproteiny chemie izolace a purifikace metabolismus MeSH
- metabolismus sacharidů MeSH
- molekulová hmotnost MeSH
- Ornithodoros imunologie MeSH
- ovce MeSH
- podjednotky proteinů chemie izolace a purifikace metabolismus MeSH
- protilátky imunologie MeSH
- transportní proteiny chemie izolace a purifikace metabolismus MeSH
- vazba proteinů MeSH
- zkřížené reakce MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- buněčné jádro MeSH
- buňky kostní dřeně cytologie chemie MeSH
- buňky stromatu chemie MeSH
- epidermální buňky MeSH
- epidermis chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- hemaglutininy analýza fyziologie metabolismus MeSH
- lidé MeSH
- prasata MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
