Salivary glands of mosquito vectors play an important role in the adaptation to haematophagy. Indeed the process of feeding on blood presents challenges to the mosquito and one of the most relevant is the demand to face the host haemostatic response to blood vessels damage. In this respect mosquito salivary glands have evolved the ability to produce and secrete substances able to counteract aspects of haemostasis such as vasoconstriction, coagulation and platelet aggregation. Anti-haemostatic activities have been identified by classical biochemical studies in the salivary gland secretions of several blood-feeding arthropods (Ribeiro J.M.C., 1987, Ann. Rev. Entomol. 32: 463-478). However, while distinct anticoagulant or vasodilator activities seem to be present in different species, apyrases appear to be the universal inhibitors of platelet aggregation in haematofagic arthropods. Apyrase is a secreted protein that hydrolyzes ATP and ADP to AMP and its function is to inhibit the ADP-induced platelet recruitment and aggregation. Apyrase activity has been found in the salivary gland extracts of different members of the Anopheles gambiae complex (Cupp E.W. et al, 1994, Am. J. Trop. Med. Hyg. 50: 235-240) but the only apyrase gene isolated and characterized so far belongs to the yellow fever mosquito Aedes aegypti (Smartt C.T. et al, 1995, Exp. Parasitol. 81: 239-248). Interestingly, the A. aegypti apyrase shows sequence similarity to the 5'-nucleotidases, a family of ubiquitous membrane-linked proteins that hydrolyze extra cellular nucleotides into membrane permeable nucleosides. It has been proposed that apyrase derived by a 5'-nucleotidase by gene duplication and subsequent divergent evolution; this probably involved the loss of a carboxyl-terminus hydrophobic domain with membrane-anchoring properties, the acquisition of tissue-specific expression and the evolution towards a new function with high adaptive role in haematophagy (Champagne D.E. et al, 1995, Proc. Natl. Acad. Sci. USA 92: 694-698). Using the Signal Sequence Trap technique (see Arcà B. et al., this meeting), that allows for the trapping of cDNAs coding for secreted and membrane-linked proteins, we have identified two different cDNA fragments, cF3 and iC6, both showing significant sequence similarity to the A. aegypti apyrase. Using specific oligonucleotide primers we were able to show by RT-PCR that cF3 is expressed only in the salivary glands of An. gambiae females, while iC6 is expressed also in males and in other female tissues. It is very likely that the cF3 cDNA fragment represents the An. gambiae apyrase while iC6 corresponds to the 5'-nucleotidase. The cF3 cDNA fragment was used as probe to screen a thoracic cDNA library from An. gambiae females and allowed for the isolation of a clone of about 2.3 kb representing the putative full-length apyrase cDNA. The deduced protein is 557 aa in length, contains at the amino-terminus a putative 22 aa long signal peptide, has a predicted molecular weight of approximately 62 kDa and contains five possible N-linked glycosylation sites. Sequence comparison to the A. aegypti apyrase shows that the two proteins, which also have very similar molecular weight, share about 51% identity and 61% similarity throughout most of their length. These observations strongly suggest that our cDNA really represents the An. gambiae homologue of the A. aegypti apyrase and its identification may be of great help in the isolation of apyrase genes from other haematophagous arthropods. We are presently screening a genomic library in the attempt to obtain the apyrase genomic clone with the main goal of isolating an An. gambiae salivary gland specific promoter that may be of great help in the design of new malaria control strategies as soon as a genetic transformation system will become available also for the malaria vector An. gambiae.

Molecular cloning of a cDNA coding for the Anopheles gambiae homologue of the Aedes aegypti apyrase / Lombardo, F.; Coluzzi, M.; Arca', Bruno. - (1998). (Intervento presentato al convegno XX Convegno SOIPA tenutosi a Roma nel 17-20 June).

Molecular cloning of a cDNA coding for the Anopheles gambiae homologue of the Aedes aegypti apyrase.

ARCA', BRUNO
1998

Abstract

Salivary glands of mosquito vectors play an important role in the adaptation to haematophagy. Indeed the process of feeding on blood presents challenges to the mosquito and one of the most relevant is the demand to face the host haemostatic response to blood vessels damage. In this respect mosquito salivary glands have evolved the ability to produce and secrete substances able to counteract aspects of haemostasis such as vasoconstriction, coagulation and platelet aggregation. Anti-haemostatic activities have been identified by classical biochemical studies in the salivary gland secretions of several blood-feeding arthropods (Ribeiro J.M.C., 1987, Ann. Rev. Entomol. 32: 463-478). However, while distinct anticoagulant or vasodilator activities seem to be present in different species, apyrases appear to be the universal inhibitors of platelet aggregation in haematofagic arthropods. Apyrase is a secreted protein that hydrolyzes ATP and ADP to AMP and its function is to inhibit the ADP-induced platelet recruitment and aggregation. Apyrase activity has been found in the salivary gland extracts of different members of the Anopheles gambiae complex (Cupp E.W. et al, 1994, Am. J. Trop. Med. Hyg. 50: 235-240) but the only apyrase gene isolated and characterized so far belongs to the yellow fever mosquito Aedes aegypti (Smartt C.T. et al, 1995, Exp. Parasitol. 81: 239-248). Interestingly, the A. aegypti apyrase shows sequence similarity to the 5'-nucleotidases, a family of ubiquitous membrane-linked proteins that hydrolyze extra cellular nucleotides into membrane permeable nucleosides. It has been proposed that apyrase derived by a 5'-nucleotidase by gene duplication and subsequent divergent evolution; this probably involved the loss of a carboxyl-terminus hydrophobic domain with membrane-anchoring properties, the acquisition of tissue-specific expression and the evolution towards a new function with high adaptive role in haematophagy (Champagne D.E. et al, 1995, Proc. Natl. Acad. Sci. USA 92: 694-698). Using the Signal Sequence Trap technique (see Arcà B. et al., this meeting), that allows for the trapping of cDNAs coding for secreted and membrane-linked proteins, we have identified two different cDNA fragments, cF3 and iC6, both showing significant sequence similarity to the A. aegypti apyrase. Using specific oligonucleotide primers we were able to show by RT-PCR that cF3 is expressed only in the salivary glands of An. gambiae females, while iC6 is expressed also in males and in other female tissues. It is very likely that the cF3 cDNA fragment represents the An. gambiae apyrase while iC6 corresponds to the 5'-nucleotidase. The cF3 cDNA fragment was used as probe to screen a thoracic cDNA library from An. gambiae females and allowed for the isolation of a clone of about 2.3 kb representing the putative full-length apyrase cDNA. The deduced protein is 557 aa in length, contains at the amino-terminus a putative 22 aa long signal peptide, has a predicted molecular weight of approximately 62 kDa and contains five possible N-linked glycosylation sites. Sequence comparison to the A. aegypti apyrase shows that the two proteins, which also have very similar molecular weight, share about 51% identity and 61% similarity throughout most of their length. These observations strongly suggest that our cDNA really represents the An. gambiae homologue of the A. aegypti apyrase and its identification may be of great help in the isolation of apyrase genes from other haematophagous arthropods. We are presently screening a genomic library in the attempt to obtain the apyrase genomic clone with the main goal of isolating an An. gambiae salivary gland specific promoter that may be of great help in the design of new malaria control strategies as soon as a genetic transformation system will become available also for the malaria vector An. gambiae.
1998
Molecular cloning of a cDNA coding for the Anopheles gambiae homologue of the Aedes aegypti apyrase / Lombardo, F.; Coluzzi, M.; Arca', Bruno. - (1998). (Intervento presentato al convegno XX Convegno SOIPA tenutosi a Roma nel 17-20 June).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/302022
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