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Fibrinogen-related proteins in ixodid ticks.

Sterba J, Dupejova J, Fiser M, Vancova M, Grubhoffer L - Parasit Vectors (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech Republic. sterbaj@paru.cas.cz

ABSTRACT

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.

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Immunoblotting of tick haemolymph proteins with anti-ficolin antibodies. Electrophoretically separated and electroblotted non-reduced haemolymph proteins from D. marginatus (lane 1), R. appendiculatus (lane 2), R. pulchellus (lane 3), and R. sanguineus (lane 4) were immunostained using rabbit anti-FCN1 H antibodies. Recombinant human ficolin 1 was used as a control (lane 5). Purified hemelipoglycoprotein from D. marginatus haemolymph, which was identified by MS as one of the recognised proteins was used as a control (lane 6). However, this protein does not contain the fibrinogen domain [3]. The same proteins as in Figure 1A were detected in D. marginatus haemolymph (36 kDa, 79/80 kDa, and 290 kDa proteins; marked with asterisks; the 79/80 kDa double-band is marked by one asterisk). In Rhipicephalus ticks haemolymphs, the 72 kDa and 290 kDa proteins were detected, but not the 55 kDa protein. Additionally, the purified hemelipoglycoprotein from D. marginatus was detected by the anti-FCN1 H antibodies. Non-reduced recombinant human ficolin 1 served as a positive control. Antibodies positively reacted with subunits of the protein (approximately 30 kDa) as well as with higher molecular weight complexes (approximately 60 kDa, 180 kDa, 250 kDa, 280 kDa).
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Figure 2: Immunoblotting of tick haemolymph proteins with anti-ficolin antibodies. Electrophoretically separated and electroblotted non-reduced haemolymph proteins from D. marginatus (lane 1), R. appendiculatus (lane 2), R. pulchellus (lane 3), and R. sanguineus (lane 4) were immunostained using rabbit anti-FCN1 H antibodies. Recombinant human ficolin 1 was used as a control (lane 5). Purified hemelipoglycoprotein from D. marginatus haemolymph, which was identified by MS as one of the recognised proteins was used as a control (lane 6). However, this protein does not contain the fibrinogen domain [3]. The same proteins as in Figure 1A were detected in D. marginatus haemolymph (36 kDa, 79/80 kDa, and 290 kDa proteins; marked with asterisks; the 79/80 kDa double-band is marked by one asterisk). In Rhipicephalus ticks haemolymphs, the 72 kDa and 290 kDa proteins were detected, but not the 55 kDa protein. Additionally, the purified hemelipoglycoprotein from D. marginatus was detected by the anti-FCN1 H antibodies. Non-reduced recombinant human ficolin 1 served as a positive control. Antibodies positively reacted with subunits of the protein (approximately 30 kDa) as well as with higher molecular weight complexes (approximately 60 kDa, 180 kDa, 250 kDa, 280 kDa).

Mentions: All four (36 kDa, 79/80 kDa, and 290 kDa) proteins were recognised in D. marginatus haemolymph by the anti-FCN1 H antibodies (Figure 2, lane 1). In the three Rhipicephalus species haemolymphs, only the 75 and 290 kDa proteins were recognised (Figure 2, lanes 2, 3, and 4). Furthermore, we performed the immunoblotting on the purified hemelipoglycoprotein from D. marginatus [4]; the purified protein was recognised by the anti-FCN1 H antibodies as well (Figure 2, lane 6). Recombinant human ficolin 1 was used as a control (Figure 2, lane 5). Similar results were obtained also using the second antibodies, anti-FCN1 S (data not shown).


Fibrinogen-related proteins in ixodid ticks.

Sterba J, Dupejova J, Fiser M, Vancova M, Grubhoffer L - Parasit Vectors (2011)

Immunoblotting of tick haemolymph proteins with anti-ficolin antibodies. Electrophoretically separated and electroblotted non-reduced haemolymph proteins from D. marginatus (lane 1), R. appendiculatus (lane 2), R. pulchellus (lane 3), and R. sanguineus (lane 4) were immunostained using rabbit anti-FCN1 H antibodies. Recombinant human ficolin 1 was used as a control (lane 5). Purified hemelipoglycoprotein from D. marginatus haemolymph, which was identified by MS as one of the recognised proteins was used as a control (lane 6). However, this protein does not contain the fibrinogen domain [3]. The same proteins as in Figure 1A were detected in D. marginatus haemolymph (36 kDa, 79/80 kDa, and 290 kDa proteins; marked with asterisks; the 79/80 kDa double-band is marked by one asterisk). In Rhipicephalus ticks haemolymphs, the 72 kDa and 290 kDa proteins were detected, but not the 55 kDa protein. Additionally, the purified hemelipoglycoprotein from D. marginatus was detected by the anti-FCN1 H antibodies. Non-reduced recombinant human ficolin 1 served as a positive control. Antibodies positively reacted with subunits of the protein (approximately 30 kDa) as well as with higher molecular weight complexes (approximately 60 kDa, 180 kDa, 250 kDa, 280 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141747&req=5

Figure 2: Immunoblotting of tick haemolymph proteins with anti-ficolin antibodies. Electrophoretically separated and electroblotted non-reduced haemolymph proteins from D. marginatus (lane 1), R. appendiculatus (lane 2), R. pulchellus (lane 3), and R. sanguineus (lane 4) were immunostained using rabbit anti-FCN1 H antibodies. Recombinant human ficolin 1 was used as a control (lane 5). Purified hemelipoglycoprotein from D. marginatus haemolymph, which was identified by MS as one of the recognised proteins was used as a control (lane 6). However, this protein does not contain the fibrinogen domain [3]. The same proteins as in Figure 1A were detected in D. marginatus haemolymph (36 kDa, 79/80 kDa, and 290 kDa proteins; marked with asterisks; the 79/80 kDa double-band is marked by one asterisk). In Rhipicephalus ticks haemolymphs, the 72 kDa and 290 kDa proteins were detected, but not the 55 kDa protein. Additionally, the purified hemelipoglycoprotein from D. marginatus was detected by the anti-FCN1 H antibodies. Non-reduced recombinant human ficolin 1 served as a positive control. Antibodies positively reacted with subunits of the protein (approximately 30 kDa) as well as with higher molecular weight complexes (approximately 60 kDa, 180 kDa, 250 kDa, 280 kDa).
Mentions: All four (36 kDa, 79/80 kDa, and 290 kDa) proteins were recognised in D. marginatus haemolymph by the anti-FCN1 H antibodies (Figure 2, lane 1). In the three Rhipicephalus species haemolymphs, only the 75 and 290 kDa proteins were recognised (Figure 2, lanes 2, 3, and 4). Furthermore, we performed the immunoblotting on the purified hemelipoglycoprotein from D. marginatus [4]; the purified protein was recognised by the anti-FCN1 H antibodies as well (Figure 2, lane 6). Recombinant human ficolin 1 was used as a control (Figure 2, lane 5). Similar results were obtained also using the second antibodies, anti-FCN1 S (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech Republic. sterbaj@paru.cas.cz

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus