title: Bradykinin release avoids high molecular weight kininogen endocytosis.
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 antipain-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.
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Igor Z
authors.initials: IZ
authors.lastname: Damasceno
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Katia R B
authors.initials: KR
authors.lastname: Melo
authors.affiliation: Programas de Biomateriais e Biotecnologia, Universidade Anhanguera de São Paulo (UNIAN SP), São Paulo, SP, Brasil.
authors.forename: Fabio D
authors.initials: FD
authors.lastname: Nascimento
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Daianne S P
authors.initials: DS
authors.lastname: Souza
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Mariana S
authors.initials: MS
authors.lastname: Araujo
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Sinval E G
authors.initials: SE
authors.lastname: Souza
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Misako U
authors.initials: MU
authors.lastname: Sampaio
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Helena B
authors.initials: HB
authors.lastname: Nader
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Ivarne L S
authors.initials: IL
authors.lastname: Tersariol
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.
authors.forename: Guacyara
authors.initials: G
authors.lastname: Motta
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000378:metabolism*; D016466:CHO Cells; D021941:Caveolae / Q000378:metabolism; D002460:Cell Line; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism
chemical_list: D019679:Kininogen, High-Molecular-Weight; D011509:Proteoglycans; D057057:Serine Proteases; D007610:Kallikreins; D001920:Bradykinin
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