Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. in the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. in CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. the H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. the anti-pain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. the present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biomat, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biotecnol, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilCNPq: CNPq 472403/2007-9FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6Web of Scienc