The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases.
Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.
Azzolini
Simone S
SS
Departamento de Bioquímica, UNIFESP-EPM, Rua Três de Maio 100, 04044-020, São Paulo, SP, Brazil.
Sasaki
Sergio D
SD
Campos
Ivan T N
IT
S Torquato
Ricardo J
RJ
Juliano
Maria A
MA
Tanaka
Aparecida S
AS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006454:Hemoglobins / Q000378:metabolism; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009117:Muscidae / Q000737:chemistry* / Q000201:enzymology / Q000276:immunology; D016133:Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000187:drug effects / Q000378:metabolism
D006454:Hemoglobins; D015842:Serine Proteinase Inhibitors; D005340:Fibrinogen; D019272:Leukocyte Elastase; D014357:Trypsin