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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

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Author(s):
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]
Total Authors: 10
Affiliation:
[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
Total Affiliations: 4
Document type: Journal article
Source: PLoS One; v. 10, n. 3 MAR 30 2015.
Web of Science Citations: 5
Abstract

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)

FAPESP's process: 13/05822-9 - Proteinase-Activated Receptors (PARs) in the dentin-pulp complex: identification, modulation and signal tranduction in caries disease
Grantee:Fábio Dupart Nascimento
Support Opportunities: Research Grants - Young Investigators Grants