title: Salivary antigen-5/CAP family members are Cu2+-dependent antioxidant enzymes that scavenge O₂₋. and inhibit collagen-induced platelet aggregation and neutrophil oxidative burst.
abstract: The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2[Symbol: see text] generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2[Symbol: see text] generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu(2+), which provides redox potential for catalytic removal of O2[Symbol: see text]. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ~100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu(2+) and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu(2+)-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland, 20892.
authors.forename: Teresa C F
authors.initials: TCF
authors.lastname: Assumpção
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland, 20892.
authors.forename: Dongying
authors.initials: D
authors.lastname: Ma
authors.affiliation: Institute of Parasitology, Academy of Sciences of the Czech Republic, Biology Centre, 37005 Ceske Budejovice, Czech Republic.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schwarz
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, NIAID, National Institutes of Health, Rockville, Maryland 20852.
authors.forename: Karine
authors.initials: K
authors.lastname: Reiter
authors.affiliation: Pathogen-Host Interface Laboratory, Department of Cell Biology, University of Brasília, 70910-900 Brasília, Brazil.
authors.forename: Jaime M
authors.initials: JM
authors.lastname: Santana
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland, 20892.
authors.forename: John F
authors.initials: JF
authors.lastname: Andersen
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland, 20892.
authors.forename: José M C
authors.initials: JMC
authors.lastname: Ribeiro
authors.affiliation: Research Technology Branch, NIAID, National Institutes of Health, Rockville, Maryland 20852.
authors.forename: Glenn
authors.initials: G
authors.lastname: Nardone
authors.affiliation: Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8391.
authors.forename: Lee L
authors.initials: LL
authors.lastname: Yu
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland, 20892. Electronic address: ifrancischetti@niaid.nih.gov.
authors.forename: Ivo M B
authors.initials: IMB
authors.lastname: Francischetti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006736:Horses; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010802:Phylogeny; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D016897:Respiratory Burst; D012469:Salivary Glands / Q000201:enzymology; D016415:Sequence Alignment; D012754:Sharks; D013455:Sulfur / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013552:Swine; D014224:Triatoma / Q000201:enzymology*
chemical_list: D000975:Antioxidants; D016166:Free Radical Scavengers; D006025:Glycosaminoglycans; D013455:Sulfur; D003300:Copper; D003094:Collagen; D006861:Hydrogen Peroxide; D010100:Oxygen
keywords: Blood; Hemostasis; Inflammation; Insect; Toxins
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