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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

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Autor(es):
Damasceno, Igor Z. [1] ; Melo, Katia R. B. [1] ; Nascimento, Fabio D. [2, 3] ; Souza, Daianne S. P. [1] ; Araujo, Mariana S. [1] ; Souza, Sinval E. G. [4] ; Sampaio, Misako U. [1] ; Nader, Helena B. [1] ; Tersariol, Ivarne L. S. [1] ; Motta, Guacyara [1]
Número total de Autores: 10
Afiliação do(s) autor(es):
[1] Univ Fed Sao Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, Sao Paulo, SP - Brazil
[2] Univ Anhanguera Sao Paulo UNIAN SP, Programa Biomat, Sao Paulo, SP - Brazil
[3] Univ Anhanguera Sao Paulo UNIAN SP, Programa Biotecnol, Sao Paulo, SP - Brazil
[4] Univ Fed Sao Paulo UNIFESP, Escola Paulista Med, Dept Biofis, Sao Paulo, SP - Brazil
Número total de Afiliações: 4
Tipo de documento: Artigo Científico
Fonte: PLoS One; v. 10, n. 3 MAR 30 2015.
Citações Web of Science: 5
Resumo

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. (AU)

Processo FAPESP: 13/05822-9 - Receptores ativados por proteases (PARs) no complexo dentina-polpa: identificação, modulação e sinalização celular durante o desenvolvimento da doença cárie
Beneficiário:Fábio Dupart Nascimento
Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
Processo FAPESP: 12/50219-6 - Estudo da interacao molecular de proteases com glicosaminoglicanos em sistema de cinetica rapida (stopped-flow):analise de dados em pre-equilibrio.
Beneficiário:Ivarne Luis dos Santos Tersariol
Modalidade de apoio: Auxílio à Pesquisa - Regular