Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extra...

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Bibliographic Details
Published in:	Experimental parasitology Vol. 106; no. 3; pp. 103 - 109
Main Authors:	Azzolini, Simone S., Santos, Joyce M.C., Souza, Adriana F., Torquato, Ricardo J.S., Hirata, Izaura Y., Andreotti, Renato, Tanaka, Aparecida S.
Format:	Journal Article
Language:	English
Published:	San Diego, CA Elsevier Inc 01-03-2004 Elsevier
Subjects:	Amino Acid Sequence Animals Base Sequence Biological and medical sciences Cattle Chromatography, Affinity Chromatography, Gel Cloning, Molecular DNA, Complementary - chemistry Fundamental and applied biological sciences. Psychology Haematobia irritans irritans Life cycle. Host-agent relationship. Pathogenesis Medically important nuisances and vectors, pests of stored products and materials: population survey and control Molecular Sequence Data Molecular Weight Muscidae Muscidae - chemistry Muscidae - enzymology Muscidae - genetics Nuisances Protozoa Sequence Analysis, Protein Serine Proteinase Inhibitors - chemistry Serine Proteinase Inhibitors - genetics Serine Proteinase Inhibitors - isolation & purification Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Muscidae Nuisance Purification Insecta Serine Parasite Haematobia irritans Characterization Arthropoda Inhibitor Ectoparasite Invertebrata Diptera
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Summary:	The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin–Sepharose has a molecular mass of 7029 Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20 nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI–Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein. Index Descriptors and Abbreviations: Haematobia irritans irritans; Serine proteinase inhibitor; Kunitz-type inhibitor; HiTI, H. i. irritans trypsin inhibitor; bovine trypsin; Human plasmin; Human neutrophil elastase; Bovine thrombin; Human factor Xa; Human factor XIIa; Human plasma kallikrein; Subtilisin A; MALDI-TOF, matrix assisted laser desorption ionization time-of-flight; cDNA, complementary deoxyribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction; TFA, trifluoroacetic acid
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0014-4894 1090-2449
DOI:	10.1016/j.exppara.2004.01.012