The first pacifastin elastase inhibitor characterized from a blood sucking animal.
Pacifastin-like protease inhibitors belong to a recent classified protease inhibitor family and they are the smallest protease inhibitors described in animals. In this work, we purified and characterized, for the first time, two neutrophil elastase inhibitors belonging to the pacifastin family from the blood sucking insect Triatoma infestans eggs. The inhibitors showed the same N-terminal sequences, molecular masses of 4257 and 4024Da by MALDI-TOF mass spectrometry and dissociation constants (Ki) for neutrophil elastase of 0.52 and 0.29nM, respectively. Using a fat body cDNA library, we cloned a pacifastin precursor containing two protease inhibitor domains similar to locust pacifastins. The first pacifastin domain translated to T. infestans purified protein, named TIPI1. Recombinant TIPI1 expressed in Pichia pastoris system showed similar inhibitory activities compared to the native inhibitor. Its precursor, called TiPP1, is mainly expressed in fat body, and it is up-regulated after blood feeding. The immune challenges of 1(a) instar T. infestans nymph with bacteria or dsRNA strongly stimulated TiPP1 expression in fat body, suggesting a possible role of TiPP1 in T. infestans immunity. This work is the first to characterize a blood feeding insect pacifastin inhibitor.
de Marco
Renato
R
Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio 100, Zip Code 04044-020, São Paulo, SP, Brazil.
Lovato
Diogo V
DV
Torquato
Ricardo J S
RJ
Clara
Renan O
RO
Buarque
Diego S
DS
Tanaka
Aparecida S
AS
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014224:Triatoma / Q000378:metabolism
D019476:Insect Proteins; D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; C106709:pacifastin; D010196:Pancreatic Elastase