Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

Autor Oliveira, Vitor Google Scholar
Garrido, Paula AG Google Scholar
Rodrigues, Claudia C. Google Scholar
Colquhoun, Alison Google Scholar
Castro, Leandro M. Google Scholar
Almeida, Paulo C. Google Scholar
Shida, Claudio S. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Camargo, Antonio CM Google Scholar
Hyslop, Stephen Google Scholar
Roberts, James L. Google Scholar
Grum-Tokars, Valerie Google Scholar
Glucksman, Marc J. Google Scholar
Ferro, Emer S. Google Scholar
Instituição Univ Cidade São Paulo
Universidade de São Paulo (USP)
Univ Mogi das Cruzes
Universidade Federal de São Paulo (UNIFESP)
Inst Butantan
Universidade Estadual de Campinas (UNICAMP)
Univ Texas
Rosalind Franklin Univ Med & Sci
Resumo The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). the constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. in the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium.. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.
Palavra-chave calcium
membrane binding
peptide metabolism
protease
thimet oligopeptidase
Idioma Inglês
Data de publicação 2005-06-01
Publicado em Febs Journal. Oxford: Blackwell Publishing, v. 272, n. 12, p. 2978-2992, 2005.
ISSN 1742-464X (Sherpa/Romeo, fator de impacto)
Publicador Blackwell Publishing
Extensão 2978-2992
Fonte http://dx.doi.org/10.1111/j.1742-4658.2005.04692.x
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000230088000008
Endereço permanente http://repositorio.unifesp.br/handle/11600/28316

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