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

Abstract

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. Abbreviations EP24.15; metalloendopeptidase 24.15; G6PD, glucose-6-phosphate dehydrogenase; PFA, paraformaldehyde.

Kinin B2Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation

“…2A, lanes 2 and 3) and secreted ( Fig. 2A, lanes 5 and 6), consistent with previous reports by other investigators Oliveira et al, 2005). Some cell-associated specific HA-EP species of lower molecular masses (60 -70 kDa) were also detected ( Fig.…”

“…On the other hand, epitopetagged EP24.15 was clearly visible, and it was found to be widely distributed in the cells with cytosolic, particulate, and secreted forms of the enzyme, which is consistent with previous observations of endogenous EP24.15 in other cells with higher endogenous enzyme expression (Crack et al, 1999;Ferro et al, 1999;Jeske et al, 2004;Oliveira et al, 2005). The particulate form of EP24.15 was localized both on the extracellular face of the plasma membrane and in endocytic vesicles, whereas no colocalization with either the ER marker calnexin or the Golgi marker GM130 was observed.…”

“…EP24.15 is a soluble enzyme that lacks any hydrophobic clusters of amino acids or a signal peptide and resides primarily in the cytosol (Shrimpton et al, 2002), but it may also be extracellularly associated with the plasma membrane (Crack et al, 1999;Jeske et al, 2004) and secreted into the media, both constitutively Oliveira et al, 2005) and after stimulation by, e.g., calcium Carreñ o et al, 2005). EP24.15 is implicated primarily in the extracellular metabolism of neuropeptides but also intracellularly in the antigen presentation of peptides generated by the proteasome Silva et al, 1999;Kim et al, 2003, Ferro et al, 2004.…”

Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

“…Although many immunologically-based studies have demonstrated EP24.15 expression on the plasma membrane, there exists some ambiguity as to the inside-outside orientation of the peptidase from a functional standpoint [7,11,1416,27,30,31]. To investigate this, we treated TG cultures with diluted trypsin, and measured EP24.15 and EP24.16 activity in cytoplasmic and plasma membrane fractions.…”

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

“…Studies using peptide agonists, at least in the case of opioid receptors, have shown that the co-endocytosed agonist is recycled back to the surface or processed in an acidic compartment, depending on the length of agonist treatment (17,53). Relatively few studies have focused on the enzymes that are responsible for post-endocytic peptide agonist degradation, although a number of peptidases have been shown to be capable of hydrolyzing neuroendocrine peptides including opioid peptides (2,45,54,55). Among them, an enzyme named enkephalinase (EC 3.4.24.11), originally thought to be solely responsible for enkephalin degradation, was soon shown to be able to degrade a number of other neuropeptides, and because it exhibited activity at neutral pH it was renamed "neutral endopeptidase" or neprilysin (6,8,56).…”