title: Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.
abstract: BACKGROUND
  Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.
  
  
  RESULTS
  Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or -170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K(D) = 0.4 nM) and to the active site of FXa (K(D) = 3.07 nM). We propose the name "Simukunin" for this novel protein.
  
  
  CONCLUSIONS
  We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.
authors.affiliation: Department of Entomology, The University of Georgia, Athens, Georgia, United States of America.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tsujimoto
authors.forename: Michail
authors.initials: M
authors.lastname: Kotsyfakis
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
authors.forename: Jai Hoon
authors.initials: JH
authors.lastname: Eum
authors.forename: Michael R
authors.initials: MR
authors.lastname: Strand
authors.forename: Donald E
authors.initials: DE
authors.lastname: Champagne
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D020134:Catalytic Domain; D056649:Cathepsin G / Q000037:antagonists & inhibitors; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007249:Inflammation; D020128:Inhibitory Concentration 50; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000737:chemistry; D012469:Salivary Glands / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000378:metabolism*; D012843:Simuliidae / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D004791:Enzyme Inhibitors; D065427:Factor Xa Inhibitors; D019476:Insect Proteins; D011994:Recombinant Proteins; C574534:SV-66 protein, Simulium vittatum; D012471:Salivary Proteins and Peptides; D056649:Cathepsin G; D010196:Pancreatic Elastase