Isolation and Characterization of the Gene Encoding a Novel Factor Xa-directed Anticoagulant from the Yellow Fever Mosquito,Aedes aegypti*
- From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900
Abstract
Mosquito salivary glands secrete a number of proteins that inhibit mammalian hemostasis and facilitate blood feeding. We have isolated the protein product and corresponding cDNA of a gene designated Anticoagulant-factor Xa(AFXa), that encodes the factor Xa (FXa)-directed anticoagulant of the yellow fever mosquito, Aedes aegypti. The protein was purified partially by cation exchange chromatography and shown by enzyme activity profiles and SDS-polyacrylamide gel electrophoresis analysis to have an M r = 54,000. The protein was purified further by preparative SDS-polyacrylamide gel electrophoresis and subjected to internal protein sequencing, and the sequence of five peptides was determined. Degenerate oligonucleotides were designed based on three of the peptide sequences, and these were used to screen an adult female salivary gland cDNA library from A. aegypti. A 1.8-kilobase pair cDNA was isolated and shown to encode a 415-amino acid conceptual translation product with a predicted molecular mass of 47.8 kDa that contains the five sequenced peptides. Hydrophobicity analysis predicts a 19-amino acid signal peptide typical for secreted proteins. Northern analysis demonstrated that AFXa is expressed only in female salivary glands. Baculovirus-expressed AFXa protein has the appropriate size and expected FXa-directed anticoagulant activity. Analysis of the primary amino acid sequence shows that the AFXa gene product has similarities to the serpin superfamily of serine protease inhibitors and may represent a novel, highly diverged member of this family.
Footnotes
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↵* This work was supported by National Institutes of Health Grant AI29746 and grants from the John D. and Catherine T. MacArthur Foundation and the Burroughs-Wellcome Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Student in the Medical Scientist Program at the College of Medicine, University of California, Irvine.
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↵§ To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, University of California, Bio. Sci. II, Rm. 3205, Irvine, CA 92697-3900. Tel.: 949-824-5930; Fax: 949-824-2814; E-mail: aajames{at}uci.edu.
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↵2 Y. Y. Umekita, R. A. Richard, and S. Liao, unpublished GenBank accession number.
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↵4 K. Stark, P. Ho, T. Schaub, and A. A. James, unpublished results.
- Abbreviations:
- FXa
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factor Xa
- AEBSF
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4-(2-aminoethyl)benzenesulfonyl fluoride
- bp
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base pair(s)
- kb
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kilobase pair(s)
- CAPS
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3-(cyclohexylamino)propanesulfonic acid
- EDTA
-
ethylenediamine tetraacetic acid
- PVDF
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polyvinylidene difluoride
- PAGE
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polyacrylamide gel electrophoresis
- SGE
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salivary gland extract
- PAI-2
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plasminogen activator inhibitor-2.
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- Received March 23, 1998.
- Revision received May 5, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
