| Full text | |
| 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 |