Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Kunitz-type serine protease inhibitor is a novel participator in anti-bacterial and anti-inflammatory responses in Japanese flounder (Paralichthys olivaceus).

Kunitz-type serine protease inhibitor (KSPI) interacts with serine protease (SP) to regulate cascade reactions in vivo and plays essential roles in innate immunity. Theoretical considerations support various functions of kspi, but further studies are required for full characterization of these functions. In this study, a KSPI molecule was identified from Japanese flounder (Paralichthys olivaceus), and was named Pokspi. The full-length cDNA sequence of Pokspi was 2810 nt, containing an open reading frame of 1527 nt, which encoded a polypeptide of 509 amino acid residues. PoKspi protein contained five conversed domains, namely, MANEC, PKD, LDLa and two Kunitz domains. Homology analysis revealed that Pokspi shared the highest similarity (83%) with its homolog in Cynoglossus semilaevis. Phylogenetic analysis indicated that Pokspi clustered with the homologs in other fishes. The mRNA transcripts of Pokspi were detected in all tested tissues, with the highest expression level in gill, followed by kidney and intestine. Its elevated expression in response to the application of Edwardsiella tarda (in vivo) and pathogen-associated molecular pattern (in vitro) suggested the involvement of Pokspi in the essential immune defense against various pathogens. Recombinant PoKspi (rPoKspi) purified from Escherichia coli exhibited not only serine protease inhibitor activities but also a broad spectrum of anti-microbial effect in a manner that was independent of any host factors. In addition, the recombinant PoKspi protein could cause the down-regulation of pro-inflammatory factors TNF-α and IL-1ß. In conclusion, Pokspi is a biologically active serine protease inhibitor endowed with anti-bacterial and anti-inflammatory property. This study provides strong evidences for understanding the innate immune defense in Japanese flounder.