Platelet-activating-factor-hydrolyzing phospholipase C in the salivary glands and saliva of the mosquito Culex quinquefasciatus
José M. C. Ribeiro* and
Ivo M. B. Francischetti
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room 126, 4 Center Drive, MSC 0425, Bethesda, MD 20892-0425, USA
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Fig. 1. Structure of platelet-activating factor (PAF) and the possible sites of hydrolysis by PAF acetyl hydrolase (1), phospholipase C (2) and phospholipase D (3). Note the ether group, which can vary in chain length.
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Fig. 2. Effect of mosquito salivary gland homogenates (SGH) on PAF-induced platelet aggregation of human platelets. (A) Platelet aggregation tracings in the absence (control, open squares) or presence of 0.5 pairs of homogenised salivary glands of Aedes aegypti (filled triangles) and Culex quinquefasciatus (circles). (B) Effect of the amount of Culex quinquefasciatus SGH preincubated with PAF for 1 min (filled circles) or 20 min (open circles) and Aedes aegypti (filled triangles) preincubated with PAF for 1 min, on PAF-induced platelet aggregation. Inhibition of the aggregation was estimated at 5 min after start of the assay. Values are means ± S.E.M. of three determinations. For more details, see Materials and methods.
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Fig. 3. Hydrolysis of PAF by Culex quinquefasciatus SGH. One pair of homogenised salivary glands was incubated with 50 µl of 100 µmol l-1 PAF in 0.1 mol l-1 ammonium acetate, pH 7.2. At the indicated times, 5 µl of the reaction mixture was diluted tenfold with 80 % methanol plus 0.1 % acetic acid and injected into the mass spectrometer. (A) Mass spectrum done immediately after mixing the substrate with the homogenate. (B) Mass spectrum at 120 min. (C–H) Disappearance of PAF-associated masses and appearance of indicated product masses. A control sample without salivary homogenates incubated for 2 h had an identical spectrum to A. The line on C was the best non-linear fit of a two-parameter hyperbolic decay curve. The lines on D–H were the best non-linear fit for a two-parameter hyperbolic curve.
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Fig. 4. Mass spectrum of the product of the hydrolysis of PAF by Culex quinquefasciatus SGH matches the spectrum of 1-O-hexadecyl-2-acetyl-rac-glycerol. (A) Product of the incubation of PAF (100 µmol l-1 final concentration) with SGH from one pair of glands (25 µl final volume) for 2 h, after which 5 µl were diluted with acid 80 % methanol and analyzed by mass spectrometry, as described in Fig. 2. (B) Mass spectrum of authentic of 1-O-hexadecyl-2-acetyl-rac-glycerol.
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Fig. 5. Specificity for PAF of the salivary phospholipase C activity of Culex quinquefasciatus SGH. PAF, enantio-PAF (E-PAF), lyso-PAF (L-PAF) and phosphatidylcholine with acyl chains of the indicated carbon lengths (C6, C10, C14 and C16) served as substrates. Assay conditions were as given in Fig. 2. Values are means ± S.E.M. of three determinations.
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Fig. 6. pH dependence of the PAF phosphorylcholine hydrolase activity of Culex quinquefasciatus SGH. The amount of PAF hydrolyzed at the indicated times (0, 30 and 60 min) of incubation and pH are indicated. Other conditions are as in Fig. 2, except that the ammonium acetate buffer was adjusted with acetic acid or ammonia to the indicated pH.
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Fig. 7. Molecular sieving chromatography of 25 pairs of homogenised salivary glands from Culex quinquefasciatus. (A) Ultraviolet absorbance of the eluate at 220 nm. Inset: Retention time of the enzymatic activity in relation to molecular mass (MW) standards. For more details, see Materials and methods. (B) PAF phosphorylcholine hydrolase activity as determined by disappearance of the masses associated with PAF. (C) PAF phosphorylcholine hydrolase activity as determined by appearance of the mass associated with the diacylglycerol product. (D) Action of the eluate fractions on PAF-induced platelet aggregation.
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Fig. 8. PAF phosphorylcholine hydrolase activity in saliva of adult female Culex quinquefasciatus. Serotonin-induced saliva from six mosquitoes was incubated with 50 µl PAF (other conditions as for Fig. 2). At various time intervals, a sample was examined by mass spectrometry. (A) Time course of PAF-associated masses (524.3 and 543.3). (B) Mass spectrum obtained immediately after mixing the reaction mixture with the mosquito saliva. (C) Spectrum after incubation for 3 h. A control sample without saliva and incubated for 3 h had a spectrum identical to that in A.
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© The Company of Biologists Ltd 2001
