Báo cáo khoa học: Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity pdf

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity Vitor Oliveira1, Paula A G Garrido2, Claudia C Rodrigues2, Alison Colquhoun2, Leandro M Castro2, Paulo C Almeida3, Claudio S Shida3, Maria A Juliano4, Luiz Juliano4, Antonio C M Camargo5, Stephen Hyslop6, James L Roberts7, Valerie Grum-Tokars8, Marc J Glucksman8 and Emer S Ferro2 ˆ ´ Laboratorio de Neurociencias, Universidade da Cidade de Sao Paulo, Brazil ˜ ˆ ´ Departamento de Biologia Celular e Desenvolvimento, Programa de Biologia Celular, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Brazil ˜ Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil ´ Departamento de Biofısica, Universidade Federal de Sao Paulo, Brazil ˜ Centro de Toxinologia Aplicada (CAT), Instituto Butantan, Brazil ˆ ´ Departamento de Farmacologia, Faculdade de Ciencias Medicas, Universidade Estadual de Campinas, Campinas, Brazil Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX, USA Midwest Proteome Center, Department of Biochemistry & Molecular Biology, Rosalind Franklin University of Medicine and Science ⁄ Chicago Medical School, USA Keywords calcium; membrane binding; peptide metabolism; protease; thimet oligopeptidase Correspondence ´ E S Ferro, Laboratorio de Comunicaca ¸ ˜o Celular, Avenida Prof Lineu Prestes1524 Sala 431, Sao Paulo, 05508-900, SP, Brazil ˜ E-mail: eferro@usp.br Note V Oliveira and P.A.G Garrido contributed equally to this work (Received February 2005, revised 24 March 2005, accepted 31 March 2005) doi:10.1111/j.1742-4658.2005.04692.x 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 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 2978 FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al The metalloendopeptidase EC 3.4.24.15 (EP24.15) represents a distinctive peptide-metabolizing enzyme with size-selectivity for peptides ranging from to 17 amino acids [1–3] EP24.15 is a monomeric, soluble, 77-kDa endopeptidase, thiol-activated, with an isoelectric point of 5.6 and a pH optimum of activity of 7.4, first isolated from the soluble fraction of the rat brain [4] This enzyme is distributed in all mammalian tissues so far examined, with high levels in testis, brain, kidney and pituitary [5] In the brain, at the electron microscopic level, EP24.15-like immunoreactivity is observed over the nuclei, cytoplasm, cross-sectioned dendrites, myelinated and unmyelinated axons, axon terminals, and in both astrocytes and oligodendrocytes [6] Recent studies have shown that EP24.15 could interact with peptides generated by the multicatalytic complex proteasome [7,8] Thus, the high concentration of the enzyme observed inside the cells could be somewhat related to the degradation of catabolic products generated by the proteasome [7,9] EP24.15 has been mainly implicated in extracellular metabolism of neuropeptides [5] Supporting an extracellular function of EP24.15, cell fractionation studies have shown that EP24.15 is present in a minor form in the particulate subcellular fractions from the central nervous tissue [10] and AtT-20 cells [11] Moreover, we previously reported that EP24.15 is secreted from distinctive cells lines, such as the rat glioma C6 [12] and mouse AtT20 anterior pituitary [13] Studies in the neuroendocrine cell line AtT-20 have shown that EP24.15 was enriched in the regulated secretory pathway [14] and could be either constitutively released in the extracellular space or upon stimulation with corticotrophin releasing hormone as well as calcium ionophore A23187 [13] However, EP24.15 secretion from AtT20 cells is partially blocked by brefeldin A or nocodazole, suggesting that this enzyme could be secreted by a pathway different from that described for other secreted proteins or neuropeptides, such as b-endorphin [13] EP24.15 secretion from glioma C6 cells is even more unusual, as it is stimulated upon incubation of the cells in the absence of calcium [12] We have previously shown that EP24.15 is released from glioma C6 cells with low calcium concentrations [12] Using these cells as a model we now show that extracellular EP24.15 activity (enzyme associated to cells and present in the medium) was not affected by the presence or absence of calcium In fact, we have shown that previously membrane-associated EP24.15 is shed into the medium in the absence of calcium Sitedirected mutagenesis on a putative calcium binding motif revealed a role for D93 in EP24.15–membrane association The location of this residue is on the FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS EP24.15 membrane association and calcium sensitivity surface, and it can bind calcium as corroborated by molecular modeling by the EP24.15 derived X-ray diffraction data Moreover, calcium was also observed to affect EP24.15 cleavage site on specific fluorogenic peptides Results The rate of shedding of EP24.15 from C6 glioma cells is inversely proportional to the calcium concentration present in the medium (Fig 1A) To analyze if this increment in EP24.15 activity into the medium was due to secretion, the following experiments were conducted: soluble EP24.15 activity was determined in the medium and subtracted from the total activity observed in the presence of cells, the latter corresponding to cells + medium activity (Fig 1B) Total extracellular EP24.15 activity (cells + media) remains the same with respect to the presence or absence of calcium (Fig 1B) However, there is a higher EP24.15 activity in the medium of cells incubated in calciumfree conditions compared with those incubated in calcium-containing medium (Fig 1B) The viability of glioma C6 cells was determined by the Trypan blue dye exclusion method, and suggested that more than 99.5 ± 5% of cells were intact In the same experiments, the activity of the cytosolic glucose-6-phosphate dehydrogenase (G6PD), an indicator of cell death, in the medium was < 0.01 ± 0.01 mU per 106 cells (n ¼ 5), from a total activity in the whole cell homogenates of 2.9 ± 0.3 mU per 106 cells (n ¼ 5), further suggesting that cells were intact after incubation in calciumfree medium The rabbit anti-EP24.15 serum recognized a single protein band of approximately 78 kDa in the crude homogenate of glioma C6 cells (Fig 2A), suggesting the specificity against this enzyme [15] Importantly, the rabbit anti-EP24.15 antiserum used here has been previously characterized not to show cross-reactivity with the related endopeptidase EP24.16 [6] EP24.15 immunostaining revealed the presence of a diffuse labeling throughout the whole cell that includes an intense nuclear labeling (Fig 2B–E) The intensity of this immunolabeling differed from cell to cell indicating a variable expression of EP24.15 in these cells No major differences in the specific immunostaining could be observed after preincubating the cells in the presence (Fig 2B) or absence (Fig 2C) of calcium The presence of Triton X100 (0.1%) during the immunocytochemical procedures (Fig 2B and C) clearly increases the nuclear labeling for EP24.15, compared to similar experiments conducted in the absence of the detergent (Fig 2D and E) These data suggest that cell 2979 EP24.15 membrane association and calcium sensitivity Fig Calcium-dependent EP24.15 released into the medium EP24.15 activity in intact cells was determined using b-lipotropin(61–69) (YGGFMTSEK; 100 lM) as substrate b-lipotropin(61–69) fragments were separated by reverse phase HPLC and the EP24.15 activity was based on the generation of [Met5]enkephalin calculated by comparing the peak areas of each of those products with the corresponding peak area of the same synthetic peptides of known concentrations The EP24.15 enzymatic activity released into the medium was determined fluorimetrically using the substrate Abz-GGFLRRV-EDDnp To discern peptidolytic activity exclusively due to EP24.15, the inhibitors N-(1-(R,S)-carboxyl2-phenylethyl)-AAF-p-amino-benzoate (CFP-AAF-pAB) and ⁄ or the dipeptide Pro–Ile were used All enzymatic determinations were conducted under linear conditions where product formation was directly proportional to enzyme concentration, and obeys zero-order kinetic parameters with < 10% of total substrate consumed during the course of the assay Results are means of three independent determinations ± SE (A) EP24.15 release into the glioma C6 cells’ medium is inversely proportional to the extracellular calcium concentration (B) Total extracellular EP24.15 activity is not affected by calcium, however, the amount of enzyme that is released in the medium is largely increased after incubating the glioma C6 cells in medium that lacks calcium permeabilization with detergent is an important issue regarding the nuclear staining for EP24.15 However, detergent and calcium removal was not sufficient to avoid intracellular EP24.15 staining (Fig 2D and E) Moreover, in control experiments, neither somatic nor nuclear labeling was observed when the anti-EP24.15 2980 V Oliveira et al antiserum was preadsorbed with the recombinant EP24.15 (100 lgỈmL)1; Fig 2C) On the other hand, immunocytochemistry performed with postfixation of the cells in the absence of Triton X100, produces a clear cell-surface immunostaining for EP24.15, with different intensities among the cells (Fig 3A) Under these latter conditions, previous incubation of the cells in calcium-free medium strongly affects the intensity of EP24.15 extracellular immunostaining (Fig 3B) These data suggest the shedding of EP24.15 from the plasma membrane after calcium removal Immunocytochemistry for electron microscopy further confirmed the presence of EP24.15 in the extracellular face of the plasma membrane of glioma C6 cells (Fig 4) We then searched for putative calcium-binding motifs on the surface of EP24.15 that could contribute to the enzyme’s membrane association At least two possible calcium-binding motifs containing an aspartic acid were identified on these structural analyses (D93 and D159), which were mutated to alanine by sitedirected mutagenesis producing the mutants D93A, D159A and the double mutant, D93 ⁄ 159A The corresponding recombinant proteins were expressed in bacteria and purified to homogeneity (Fig 5A) Western blot analyses show that such point mutations have no effect on antiserum recognition (Fig 5B) The following experiments were performed in an attempt to correlate a possible effect of the above point mutations with the calcium-dependent EP24.15 membrane association The homogeneously purified EP24.15 recombinant proteins (wild-type or mutant) were incubated with glioma C6 cells and the putative membrane binding was analyzed by confocal microscopy Incubation of either wild-type EP24.15 or the D159A mutant with the glioma C6 cells produced a clear increment in the immunoreactivity of EP24.15 associated with the extracellular surface of these cells (Fig 6A and B) when compared with control performed without adding any of the recombinant EP24.15 (Fig 6E) On the other hand, incubation of the glioma C6 cells with either D93A or D93 ⁄ 159A mutated proteins was not able to produce a similar increment in the extracellular immunoreactivity related to EP24.15 (Fig 6C and D) Control experiments under these conditions show that without the addition of EP24.15 the basal immunoreactivity was greatly reduced (Fig 6E) Taken together, these data suggest the D93 residue of EP24.15 within the putative calcium-binding motif 89SPNKD93 is a possible mediator of enzyme–membrane association To examine a structure–function correlate of the D93–membrane association and the location of the residue within the three-dimensional realm of the enzyme, FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al EP24.15 membrane association and calcium sensitivity Fig EP24.15 immunoreactivity in glioma C6 cells EP24.15 presence in glioma C6 cells was further confirmed by immunochemical reactions (A) The antiserum raised against the rat testis recombinant EP24.15 recognized in the whole homogenate of glioma C6 cells a single protein band corresponding to the expected Mr of EP24.15 For details see Experimental procedures (B–E) Prior to each immunocytochemistry experiment the DMEM culture medium was removed and cells were rinsed three times with fresh NaCl ⁄ Pi, with (B, D) or without (C, E) 1.2 mM of calcium, and incubated for 30 in 4% formaldehyde, pH 7.4 After three cycles of NaCl ⁄ Pi washing (15 each) cells were incubated for h in the presence of rabbit anti-EP24.15 serum (1 : 4000) diluted in NaCl ⁄ Pi containing 3% normal goat serum, 1% BSA, in the presence (B, C) or absence (D, E) of 0.1% of Triton X100 Labeled cells were examined under a Zeiss laser confocal microscope (CLSM 410) equipped with an Axiovert 100 inverted microscope and an Argon ⁄ Krypton laser Cy3-tagged molecules were excited at a wavelength of 568 nm Images were acquired sequentially as single transcellular optical sections and averaged over 32 scans per frame They were then processed using the Carl Zeiss CLSM software (version 3.1) and stored on Jazz disks for further retrieval and editing Final composites were prepared using Adobe PHOTOSHOP without modifying the spectral characteristics of the original signal Control experiments were performed as described; no specific cell labeling could be observed (data not shown) The data presented are representative of three experiments performed independently and exhibiting similar results a hypothetical intermolecular interaction using calcium as a ligand was performed (Fig 7) The calcium is modeled in green and is hydrogen bonded to two amino acids The first amino acid is D93, the object of study in this work Using an ensemble of coordinates of well studied Asp–calcium interactions, the carboxy groups of the Asp are within hydrogen bonding distance (2.5–3 A) for calcium The coordinates for Fig were obtained from the crystal structure of thimet oligopeptidase deposited at the Research Collaboratory FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS for Structural Bioinformatics (Protein Data Bank ID #1S4B) The figure was drawn with the computer modeling program spock and then subsequently rendered using raster 3d [16] To ensure that electrostatics were properly accounted for, calculations were performed and slightly negative potentials were obtained for the D93 Furthermore, we investigated the calcium effects on EP24.15 secondary structure, enzymatic activity and substrate specificity Enzymatic activity of the mutated 2981 EP24.15 membrane association and calcium sensitivity V Oliveira et al Fig The effects of calcium on the extracellular EP24.15 immunoreactivity in glioma C6 cells (A, B) To observe exclusively the extracellular EP24.15 immunoreactivity in glioma C6 cells, cells were incubated for 15 at °C in the presence of rabbit anti-EP24.15 serum (1 : 500), diluted in NaCl ⁄ Pi containing 3% normal goat serum and 1% BSA, prior to fixation with 4% formaldehyde pH 7.4 Labeled cells were examined under a Zeiss laser confocal microscope (CLSM 410) equipped with an Axiovert 100 inverted microscope and an Argon ⁄ Krypton laser Cy3-tagged molecules were excited at a wavelength of 568 nm Images were acquired sequentially as single transcellular optical sections and averaged over 32 scans per frame They were then processed using the Carl Zeiss CLSM software (version 3.1) and stored on Jazz disks for further retrieval and editing Final composites were prepared using Adobe PHOTOSHOP without modifying the spectral characteristics of the original signal Immunocytochemistry reactions were conducted after incubation of the glioma C6 cells in the presence (A) or absence (B) of calcium for h Note that cells previously incubated in medium lacking calcium (B) show a reduced extracellular labeling for EP24.15 compared to cells incubated in the presence of calcium (A) The intensity of the immunostaining varies from cell to cell but is consistently stronger in cells previously incubated in the presence of calcium compared to those incubated in the absence of calcium (1) Confocal regular image; (2) nomarski; (3) glow scale The immunolabeling intensity is shown in the graphic (intensity grows from green to red) No immunolabeling is observed after preabsorbing the anti-EP24.15 serum with recombinant EP24.15 (100 lgỈmL)1; data not shown) Fig Extracellular EP24.15 immunogold labeling observed by electron microscopy Cells incubated in the presence of calcium were processed for electron microscopy using a pre-embedding procedure as described (A) Lower magnification of an entire glioma C6 cell observed in the electron microscope and immunolabeled at the extracellular surface for EP24.15 (arrows) (B) Higher magnification of the cell shown in (A) immunolabeled for EP24.15 (arrows) Scale bar: A, 140 nm; B, 500 nm proteins either in the presence or absence of calcium was not different from the wild-type EP24.15 (Fig 5C) The superimposable CD spectra indicates 2982 that there are no gross perturbations in expression of the mutant proteins with respect to folding CD spectra of wild-type EP24.15 and mutants without FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al A WT EP24.15 membrane association and calcium sensitivity 93 159 93/159 78kDa B 93 159 93/159 78kDa C 2+ - Ca 2+ + Ca UAF/min WT D93A D159A D93/159A EP24.15 Fig SDS ⁄ PAGE and western blot of EP24.15 recombinant proteins (A) SDS ⁄ PAGE stained with Comassie blue shows the homogeneity of the recombinant proteins (5 lgỈlane)1) EP24.15 wild-type (WT), D93A (93), D159A (159) and D93 ⁄ 159A (93 ⁄ 159) after the affinity purification as described (B) Western blotting of the recombinant proteins (0.5 lgỈlane)1) EP24.15 wild type (WT), D93A (93), D159A (159) and D93 ⁄ 159A (93 ⁄ 159) using the anti-EP24.15 serum Note that none of the mutations affected the specificity of the antiserum for the EP24.15 (C) Comparative enzymatic activity of EP24.15 WT and mutants D93A, D159A and D93 ⁄ 159A measured with the fluorogenic substrate QFS in the absence and in the presence of mM CaCl2 Note that calcium slightly increases EP24.15 enzymatic activity in these assays smoothing and curve fitting shows a predominance of a-helical structures (Table 1; Fig 8A–D), which is similar to the estimated consensus secondary structure prediction obtained from different algorithms (http:// npsa-pbil.ibcp.fr), and more importantly, the structure of the protein solved by X-ray crystallography [17] Far UV-CD analysis indicated that the small differences observed among the wild-type and mutated EP24.15 falls within the experimental error determined using three different preparations of the enzyme (data not shown) The inter-experimental errors are  4% However, calcium (2.2 mm) addition reduced the a-helical content with a concomitant increase in the b-strand, turn and unordered structure of EP24.15 FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS (Table 1); similar results were observed in the near UV-CD spectra, collected either in the presence or in the absence of calcium (data not shown) These data clearly show that calcium affects EP24.15 secondary structure The D93A mutation alone or in combination with D159A that prevents EP24.15 membrane association seems insufficient to completely prevent the calcium changes induced in the enzyme secondary structure (Table 1), suggesting that structural changes may not correlate entirely with the enzyme membrane association With regards to substrate cleavage specificity, the gradual increase in the calcium concentration from to 50 mm raised the preference of the wild-type EP24.15 for the cleavage at the R–R bond in the ortho-aminobenzoic acid (Abz)-GGFLRRVQ-EDDnp substrate from 64% (0 mm Ca+2), to 79% (50 mm Ca+2) with a corresponding decrease in the cleavage at the L–R peptide bond from 36% (0 mm Ca+2) to 21% (50 mm Ca+2; Table 2) This variation is even more pronounced with the Abz-GGFLRRDQ-EDDnp substrate, which yielded cleavages of 63% and 37% at the L–R and R–R bonds, respectively, in the absence of Ca2+ In the presence of 50 mm of Ca2+ the preferred hydrolyzed peptide bond changes to 26% and 74% for the cleavage at the L–R and R–R bonds, respectively (Table 2) Moreover, after a mm calcium increment, the kcat ⁄ Km (lm)1Ỉs)1) ratio of both AbzGGFLRRVQ-EDDnp and Abz-GGFLRRDQ-EDDnp started to change (Table 2) These data suggest that in the presence of high calcium concentrations, as found in specific cellular microenvironments [18], EP24.15 substrate specificity could be changed Mutations of EP24.15 either at D93 or D159 have no major effects on the ratio of Abz-GGFLRRDQ-EDDnp peptide bond cleavage after calcium addition (data not shown) Discussion The most important finding of this report is that calcium regulates the membrane association, secondary structure and substrate specificity of the endo-oligopeptidase EP24.15 Site-directed mutagenesis indicated that residue D93 plays an important role in this calcium-dependent membrane association of EP24.15 in glioma C6 cells (Fig 6) This finding is consistent with the position of the residue on the surface of the enzyme as modeled on the X-ray structure (Fig 7), although we have been unable to demonstrate the direct interaction of calcium with the enzyme Moreover, biochemical analyses also indicated that calcium affects EP24.15 secondary structure (Fig 8) and 2983 EP24.15 membrane association and calcium sensitivity V Oliveira et al Fig Immunocytochemical staining of glioma C6 cells after incubation with the recombinant EP24.15 proteins Glioma C6 cells were preincubated at °C in the presence (A–D) or absence (E) of lg of either EP24.15 wild-type (A), D159A (B), D93A (C) or D93 ⁄ 159A (D), for 30 min, in DMEM containing BSA 0.05% The medium was then removed and cells were rinsed three times with DMEM and incubated at °C for 30 in the presence of the anti-EP24.15 serum (1 : 500) The excess of primary antiserum was removed rinsing the cells three times in mL of NaCl ⁄ Pi containing 5% BSA, before fixing the cells in PFA 4% The secondary anti-(rabbit Cy3) Ig (1 : 250; Sigma) was used to develop the immunoreaction Note that the intensity of the EP24.15-related immunostaining is reduced for either the D93A or D93 ⁄ 159A proteins compared to the wild-type and D159A Fig Molecular modeling of the surface EP24.15 D93 residue complexed with a calcium ion On the left a diagram of the side chain of D93 and a bound calcium (gray sphere) mapped onto the surface of the recently solved EP24.15 structure ([40] PDB ID #1S4B) On the right is a magnified view focusing on the D93 side chain and interaction with calcium The EP24.15 model with the calcium ion was energy minimized using Molecular Operating Environment software, and the figure was generated using SPOCK 2984 FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al EP24.15 membrane association and calcium sensitivity Table Calcium effects on the secondary structure of EP24.15 a-Helix TBS WT 42.4% D93A 44.3% D159A 57.5% D93 ⁄ 159A 50.3% TBS + 2.2 mM Ca2+ WT 38.7% D93A 40.2% D159A 49.1% D93 ⁄ 159A 47.1% b-Strand Turn Unordered 13.9% 14.7% 10.6% 12.3% 14.3% 12.6% 11.4% 12.6% 26.9% 25.8% 22.3% 24.5% 15.6% 15.9% 12.7% 14.6% 14.4% 13.5% 12.8% 12.3% 27.6% 27.2% 25.1% 25.6% changes the enzyme substrate specificity (Table 2) These data suggest that calcium is an important mediator of EP24.15 biological function It has been previously shown that in the brain EP24.15 is present both in neurons and glial cells [5,6] Glial cells are at least 10 times more abundant than neurons in the brain [19] and during the past years they have been recognized to be more than a connective tissue responsible for neuronal support [20] Glial cells are now identified as excitable cells capable of modulating neuronal stimulus [19] As for neurons, glial cells can also produce growth factors, pro-hormone processing enzymes [21], receptors [22], neurotransmitters [19] and neuropeptides [23] However, in contrast to neurons, which process peptides within the secretory pathway, glial cells secrete immature peptide precursors [24,25] Glial post-translational processing of opioid peptides precursor (e.g proenkephalin) is therefore thought to occur in the extracellular environment, as demonstrated by the presence of protease inhibitors in the cultured medium of striatal astrocytes that prevented Met-enkephalin generation from secreted proenkephalin [25–27] Therefore, the present results suggest that in physiological conditions EP24.15 would be able to associate with the extracellular surface of glial cells and could participate in the metabolism of a specific set of peptides released by these cells and ⁄ or from neurons Thus, further characterization of peptide-processing enzymes such as EP24.15 in the extracellular milieu of glial cells is of neurobiological significance Fig Far UV-CD spectra of the EP24.15 WT (A), D93A (B), D159A (C) and D93 ⁄ 159A (D) mutants both in the absence (n) or in the presence of CaCl2 mM (h) Note that calcium addition reduces the a-helical content of all EP24.15s analyzed (Table 1) FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS 2985 EP24.15 membrane association and calcium sensitivity Table Influence of calcium concentration on EP24.15 peptide bond cleavage Cleaved bond (%) Abz-GGFLRRVQ-EDDnp Abz-GGFLRRDQ-EDDnp [Ca2+] (mM) L–Rb R–Rb kcat ⁄ Kma (lM)1.s)1) L–Rb R–Rb kcat ⁄ Kma (lM)1.s)1) 0.5 10 50 36 – – – 31 28 21 64 – – – 69 72 79 2.0 – – – 1.5 1.2 0.8 63 62 61 56 52 45 26 37 38 39 44 48 55 74 1.3 1.3 1.3 1.3 1.4 1.3 0.7 a The SD were < 5% for any of the obtained kinetic parameters b In three independent assays conduced in triplicate the errors obtained in the percentage of hydrolyzed peptide bond was less than 3% In a previous study we reported that EP24.15 secretion from glioma C6 cells was increased linearly by reduced extracellular calcium concentrations [12] These data were paradoxical to many other secretory systems in which extracellular calcium is required for secretion [28]; therefore, the mechanism behind this calcium effect on EP24.15 secretion was investigated further It is known that extracellular calcium is not required for constitutive protein secretion [29] and reduces the release of parathyroid hormone [28] Biochemical observations shown here indicated that extracellular calcium concentrations could not change the overall rate of peptide metabolism from intact glioma C6 cells in culture (Fig 1) These data suggest that the overall extracellular EP24.15 enzymatic activity was not affected by altered calcium concentrations Consequently, we conclude that calcium removal was not really affecting EP24.15 secretion from glioma C6 cells Another possibility was that low extracellular calcium concentration compromised the EP24.15 association with the E face of the plasma membrane The presence of EP24.15 associated with the E face of the plasma membrane in glioma C6 cells was confirmed by immunocytochemistry experiments both at the light and electron microscopy levels Indeed, immunocytochemistry analyses shown here suggested that EP24.15 extracellular immunoreactivity is deeply affected when extracellular calcium is removed from glioma C6 cells Thus, it seems clear that a deficiency in extracellular calcium affects the shedding of EP24.15 from the plasma membrane of glioma C6 cells into the medium, suggesting that EP24.15–membrane association within cells can be dynamically modulated While further investigation 2986 V Oliveira et al would be necessary, this mechanism could be relevant for the intracellular traffic and secretion of EP24.15 [13,30], since it could dynamically locate the enzyme in specific cell membranes, such as the endoplasmic reticulum [6], Golgi apparatus, secretory vesicles [14] and plasma membrane [11] Interestingly, EP24.15 contains neither a leader peptide sequence nor any other hydrophobic domains to mediate its interaction with biological membranes [31] and the mechanism responsible for the enzyme membrane association in neuroendocrine cells remains unknown [6,11] The EP24.15 crystal structure was recently solved [17] showing that the enzyme surface contains 80% of all charged residues Among these amino acid residues D93 and D159 were investigated as possible mediators of EP24.15–membrane association The D93 amino acid residue is located within the EP24.15 89SPNKD93 sequence, which recalls the calcium-binding motif DXSXS previously described [32] On the other hand, D159 residue is the EP24.15 corresponding metal divalent-binding site residue identified in the homologue endopeptidase EP24.16; both D93 and D159 residues are located at the external surface of the enzyme [17,33] The D93A or D93 ⁄ 159A double mutations were shown to affect the calciumdependent association of EP24.15 to the plasma membrane of glioma C6 cells, while the D159A point mutation had no such effect The molecular modeling of the interaction of calcium and D93 on the surface of the enzyme has the correct electrostatic potential Additionally, the calcium is far enough from the active site zinc that no direct interaction can be postulated for direct effects on catalysis Changes appear to be transduced through changes in secondary structure Circular dichroism experiments reported here clearly shows that calcium affects EP24.15 secondary structure, reducing the a-helical content by 3–8% It is well known that calcium affects the conformational structure of several proteins [34–37], and that could provide a possible mechanistic explanation for the data shown above The D93A or D93 ⁄ 159A mutations that strongly affected EP24.15 membrane association were not sufficient to prevent the secondary structure alterations of EP24.15 induced by calcium Hence, it is plausible to suggest that the structural changes induced by calcium on EP24.15 secondary structure are not correlated with the membrane association Therefore, the exact role of D93 in EP24.15 membrane association remains unknown A possible explanation that needs further attention is that D93 mediates the interaction of EP24.15 with a yet unidentified membrane protein Molecular modeling corroborates that the residue is on the surface FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al A previous report demonstrated the influence of calcium and other divalent cations activating EP24.15 enzyme activity, in a substrate dependent manner [38] consistent with our data Another recent study has also shown the influence of calcium on the acid limb of a pH-dependence activity curve of EP24.15 [39] Fluorogenic substrates have also been used previously to show the influence of different salts on the EP24.15 activity [40] Our present data confirm these previous studies as calcium affected the kcat ⁄ KM ratio of at least two fluorogenic substrates hydrolyzed by EP24.15 (Table 2) In addition, we have shown here that calcium affect the ratio of peptide bond (L–R ⁄ R–R) cleavage observed with the fluorogenic substrates AbzGGFLRRDQ-EDDnp and Abz-GGFLRRVQ-EDDnp assayed These effects were more pronounced using the substrate Abz-GGFLRRDQ-EDDnp, which contains an aspartic acid residue that could interact directly with calcium However, the effect was also verified with the substrate Abz-GGFLRRVQ-EDDnp that does not contain any negatively charged residues to bind calcium directly, thus suggesting that the structural changes induced by calcium on EP24.15 secondary structure could modulate its substrate specificity, even altering the cleavage site on specific peptides Moreover, the results obtained with the substrate AbzGGFLRRDQ-EDDnp seem to indicate the presence of a positive residue at the S3¢ subsite Thus, if the kcat ⁄ KM ratio obtained from the kinetics with the substrate Abz-GGFLRRDQ-EDDnp containing a negative change at P3¢ is decomposed into the cleavage sites, it is possible to verify that the cleavage at the R–R bond was activated while the cleavage at the L–R bond was inhibited at higher calcium concentrations Similar analyses using the substrate AbzGGFLRRVQ-EDDnp, which that does not contain a negative change at P3¢, shows inhibition of both R–R and L–R cleaved sites These observations suggest that EP24.15 contains a positively charged residue at the S3¢ subsite This is in agreement with previously published reports [3,40], and could be of importance in the development of new specific substrates and ⁄ or inhibitors for the enzyme, as the detailed architecture of the enzyme and its interactions with substrates are deciphered The mutations D93A and D159A neither prevented the effect of calcium ions on the cleaved peptide bond ratio (L–R ⁄ R–R) nor changed the kcat ⁄ KM ratio for these substrates (data not shown) These mutations that could not prevent the structural changes induced by calcium on EP24.15 structure as observed with the far UV-CD assays suggest the possible existence of other calcium binding site(s) in FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS EP24.15 membrane association and calcium sensitivity the EP24.15 structure, which have not yet been described In conclusion, the present report provides evidence that EP24.15 is able to associate to the extracellular face of the plasma membrane in a calcium dependent fashion Calcium was also shown to change the secondary structure by reduction of a-helical content and to change the substrate specificity of EP24.15 Together, these new findings are useful to understand the complex organization of peptide metabolism within cells mediated by divalent cation by altering and regulating enzyme localization within subcellular compartments in the cell Experimental procedures Cell culture Glioma C6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen), 50 mL)1 penicillin G, and 50 lgỈmL)1 streptomycin sulfate, as previously described [12] The cells were maintained in 12-well plates (5.2 · 105 cellsỈcm)2 density of saturation), at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air Prior to the experiments, the culture medium from 60– 80% confluent cells was removed and cells were rinsed three times with mL Henk’s buffer (136 mm NaCl, 3.8 mm KCl, 1.2 mm CaCl2, 0.8 mm MgSO4, 1.1 mm Na2HPO4, 0.48 mm KH2PO4, 2.0 mm NHCO3, gỈL)1 d-glucose, final pH 7.4) equilibrated at 37 °C For the calcium-free experiments, cells were previously incubated for h in fresh NaCl ⁄ Pi (0.2 m phosphate buffer pH 7.4, containing 0.15 m NaCl), at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air Determination of EP24.15 activity of intact glioma C6 cells by HPLC Total extracellular EP24.15 activity was determined using b-lipotropin(61–69) (YGGFMTSEK) [12] Experiments were conducted in 12-well plates ( 5.2 · 105 cellsỈcm)2) containing 0.5 mLỈwell)1 Henk’s media The extent of hydrolysis was consistently < 10% during all the determinations The enzymatic reactions were terminated by addition of lL 8% H3PO4 Peptide fragments were separated by reverse phase HPLC using a C18 lBondapak column (4.6 · 250 mm; Millipore Corp., Danvers, MA, USA) with a linear gradient of 5–35% acetonitrile in 0.085% H3PO4 for 15 at a flow rate of mLỈmin)1 The rate of the reaction was evaluated by determining the amount of [Met5]enkephalin formed from b-lipotropin(61–69) Absorbance was monitored at a wavelength of 214 nm [Met5]enkephalin concentrations were calculated by 2987 EP24.15 membrane association and calcium sensitivity comparing the peak areas of each of those products with the corresponding peak area of the same synthetic peptides of known concentrations The EP24.15 enzymatic activity released into the medium was determined fluorimetrically using the substrate Abz-GGFLRRV-EDDnp (QF7) [16] To discern peptidolytic activity exclusively due to EP24.15, the inhibitors N-(1-(R,S)-carboxyl-2-phenylethyl)-AAF-pamino-benzoate (CFP-AAF-pAB) and ⁄ or the dipeptide Pro–Ile were used All enzymatic determinations were conducted under linear conditions where product formation was directly proportional to enzyme concentration, with < 10% of total substrate consumed during the course of the assay The Abz-GGFL fluorescent product was used as a calibration standard Enzyme assays were conducted in a final volume of 100 lL containing 10–50 lL cell extract ⁄ media, 13 lm QF7, mm 2-mercaptoethanol, and TBS (0.025 m Tris ⁄ HCl pH 7.4, 0.125 m NaCl) After 30 min, the reactions were terminated by addition of 1.9 mL 80 mm sodium formate pH 4.0 One milli-unit EP24.15 activity is defined as the amount of enzyme (inhibited by CFPAAF-pAB but not by Pro–Ile), able to hydrolyze nmol QF7Ỉmin)1ỈmL)1, at 37 °C pH 7.5, in TBS containing mm b-mercaptoethanol Results were expressed as the mean ± SD of five independent determinations Determination of soluble G6PD enzyme activity Incubation media from C6 cells were centrifuged at 14 000 g for and filtered through a 0.2-lm filter (Millipore), prior to determination of the extracellular EP24.15 and G6PD enzyme activities Intact C6 cells from each well ( 5.2 · 105 cellsỈcm)2) were re-suspended and homogenized in mL ice cold TBS using a Potter–Elvehjem homogenizer (10 · 30 s at 900 r.p.m) The samples were then centrifuged (105 000 g, 60 min) and the supernatant (cytosolic fraction) was used to determine both EP24.15 and G6PD enzyme activities G6PD enzyme activity was determined as previously described [13], in a mL (final volume) reaction mixture of 0.1 m Tris ⁄ HCl pH 8.0, 0.1 mm glucose-6-phosphate, mm NADP+, and an amount of sample able to alter 0.05–0.1 units of absorbance at 340 nmỈmin)1 at 25 °C, in the linear portion of the enzyme kinetics One milli-unit of G6PD was defined as the amount of enzyme able to increase 0.01 units of absorbance at 340 nmỈmin)1 at 25 °C Results were expressed as the mean ± SD of five independent determinations Trypan blue exclusion assay After experiments, cells were rinsed twice with Henk’s media and treated with 0.2% Trypan blue solution in NaCl ⁄ Pi for Dye excess was removed from the cell supernatant after a brief centrifugation (1000 g, min), and cells were counted using a Neubauer chamber (Hirschmann) Cells that did not incorporate the dye 2988 V Oliveira et al were considered intact Results were expressed as the mean ± SD of three independent determinations Western blots After electrophoretic separation by SDS ⁄ PAGE on an 8% acrylamide gel, proteins from the glioma C6 cells crude homogenate (50 lg per lane) were transferred to nitrocellulose membranes and western blots were performed using the rabbit anti-EP24.15 serum (1 : 2000), diluted in NaCl ⁄ Pi containing 5% nonfat dry milk (w ⁄ v) as described previously [18] For controls, the antiserum was replaced by serum preadsorbed with 100 lgỈmL)1 of recombinant (EP24.15) After h incubation at room temperature in the presence of the primary antiserum, the membranes were rinsed with TBS containing 0.1% Tween 20 and 5% nonfat milk and incubated with peroxidase-conjugated goat antirabbit IgGs (1 : 5000; GE Healthcare, Amersham, UK) for h Immunoreactive bands on the nitrocellulose membranes were visualized using an ECL kit (GE Healthcare) according to the manufacturer’s instructions Immunocytochemistry for light and electron microscopy C6 glioma cells were cultured as described above under cover slips For the calcium-free experiments cells were previously incubated for h in fresh NaCl ⁄ Pi, at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air Prior to each immunocytochemistry experiment, culture medium was removed and cells were rinsed three times with fresh NaCl ⁄ Pi and incubated for 30 in 4% formaldehyde pH 7.4 After three cycles of NaCl ⁄ Pi washing (15 each) cells were incubated h in the presence of rabbit anti-EP24.15 serum (1 : 4000) diluted in NaCl ⁄ Pi containing 3% normal goat serum, 1% BSA and 0.01% or 0.1% of Triton X100 Triton X100 was omitted in some experiments as indicated in the figure legend To analyze the extracellular EP24.15 immunoreactivity, experiments were conducted by incubating the cells for 15 at °C in the presence of rabbit anti-EP24.15 serum (1 : 500), diluted in NaCl ⁄ Pi containing 3% normal goat serum, 1% BSA, prior to the addition of the 4% formaldehyde, pH 7.4 After three cycles of 15 wash in NaCl ⁄ Pi, anti-(rabbit Cy3-conjugated) Ig (1 : 250; Sigma) diluted in NaCl ⁄ Pi containing 0.01% Triton X100, 3% normal goat serum and 1% BSA was added to the cells, and incubated for h at room temperature Controls for the EP24.15 immunocytochemical reaction consisted of both preabsorbing the anti-EP24.15 serum with recombinant EP24.15 (100 lgỈmL)1) and incubation of the preimmune serum (1 : 4000) instead of the anti-EP24.15 serum Following several cycles of washing in NaCl ⁄ Pi, the cover slips were mounted over slide glass using glycerol The immunofluorescent staining was observed and photographed on FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS V Oliveira et al either a microscope equipped for epifluorescence with the corresponding filters (data not shown), or on a confocal microscope as described below Labeled cells were examined under a Zeiss laser confocal microscope (CLSM 410) equipped with an Axiovert 100 inverted microscope and an Argon ⁄ Krypton laser Cy3-tagged molecules were excited at a wavelength of 568 nm Images were acquired sequentially as single transcellular optical sections and averaged over 32 scans per frame They were then processed using the Carl Zeiss clsm software (version 3.1) and stored for further retrieval and editing Final composites were prepared using Adobe’s photoshop without modifying the spectral characteristics of the original signal For electron microscopy, cells were processed using a pre-embedding procedure [6] Briefly, cells were fixed at °C for h in 3.75% acrolein and 4% paraformaldehyde (PFA) in 0.1 m phosphate buffer, pH 7.4 After that, cells were washed extensively in phosphate buffer, reacted with sodium borohydride, washed again in phosphate buffer, and cryoprotected for 30 by immersion in a mixture of 6% glycerol and 25% sucrose in 0.05 m phosphate buffer They were then rapidly frozen in isopentane at )80 °C, transferred to liquid nitrogen for min, and thawed in phosphate buffer at room temperature Thawed cells were incubated for 30 in TBS containing 3% of normal goat serum, and then for 18 h at °C in rabbit anti-EP24.15 serum diluted : 1000 in TBS containing 0.5% normal goat serum Cells were rinsed in 0.01 m NaCl ⁄ Pi (0.01 m phosphate buffer pH 7.4, containing 0.9% NaCl), incubated for h in a : 50 dilution of colloidal gold (1 nm)-conjugated goat anti-rabbit IgGs (Amersham, Arlington Heights, IL, USA) diluted in 0.01 m NaCl ⁄ Pi containing 0.2% gelatin and 0.8% BSA, washed again in 0.01 m NaCl ⁄ Pi, and fixed for 10 in 2% glutaraldehyde in 0.01 m NaCl ⁄ Pi After several washes in 0.2 m citrate buffer, pH 7.4, the immunogold was silver-enhanced by incubating the sections for with IntenSE M silver solution (Amersham) The reaction was stopped by washing in citrate buffer, and postfixed in 2% osmium tetroxide in phosphate buffer for 40 min, dehydrated in graded ethanols and flat-embedded in Polybed 812 (Polysciences, Inc., Warrington, Pennsylvania, USA) Samples were blocked off from the bottom of the plate, glued to the tip of a polymerized Polybed chuck, and cut at 80-nm thickness on an ultramicrotome Ultrathin sections were counterstained with lead citrate and uranyl acetate and examined with a JEOL 1010 TEM Negatives from electron microscopic photomicrographs were scanned at 1200 dpi resolution on an AGFA Duoscan T1200 scanner and final composites were prepared using Adobe’s photoshop without modifying the original data Site-direct mutagenesis and protein expression Double-stranded site-directed mutagenesis of rat EP24.15 was performed on a pGEX-4t2 (Amersham-Pharmacia FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS EP24.15 membrane association and calcium sensitivity Biotech Inc.) using the protocols described by the manufactory of the Quick-change Site-directed mutagenesis kit (Stratagene, Inc) Oligonucleotide primers 5¢-CGTGTCTCCGAA CAAGGCAATCCGCGCAGC-¢3 (sense) and 5¢-GCTGCG CGGATTGCCTTGTTCGGAGACACG-3¢ (antisense) and 5¢-CCACTTACCTCAGGCAACACAGGAGAAGATCAAG3¢ (sense) and 5¢-CTTGATCTTCTCCTGTGTTGCCTG AGGTAAGTGG-3¢ (antisense) were used to introduce the D93A and D159A point mutations on EP24.15, respectively; double mutant was obtained from the D93A mutated cDNA using the D159A oligos The plasmid DNA was purified (Mini-Prep, Promega Corp., Madison, WI, USA) and mutations screened by automatic DNA sequencing, using a MegaBace machine (GE Healthcare) Plasmid DNA containing the desired mutation was purified (Mini-Prep, Promega Corp.) and transformed into electrocompetent DH5a bacterial cells and plated overnight on plates containing ampicillin to yield single colonies Expression and purification of the mutant proteins for biochemical characterization were performed as described [15] Purification to homogeneity was assessed by SDS ⁄ PAGE, and protein was quantified by the Bradford assay [41] Yields of expressed protein were similar for all of the mutations and proper folding was verified by CD (described below) Aliquots were stored at )80 °C for subsequent studies CD Circular dichroism experiments were performed in a Jobin Yvon CD6 spectropolarimeter Calibration was made using d-10-camphosulfonic acid CD spectra were collected in the wavelength range of 195–260 nm (far UV-CD) or 240–350 nm (near UV-CD) at 0.5 nm resolution, s response, eight scans, 0.01 or 0.02 cm path length cells, and temperature 20–22 °C Ellipticity is reported as mean residue molar ellipticity [h] (deg cm2Ỉdmol)1) The control baseline was obtained with solvent and all the components (TBS) without the proteins All the data were obtained with three different solutions of the proteins Secondary structure estimation of the proteins was performed using data in the wavelength range of 195–260 nm using SELCON algorithm [42] Kinetic assays The hydrolysis of the quenched fluorescence peptide substrates at 37 °C in TBS was followed by measuring the fluorescence at kem ¼ 420 nm and kex ¼ 320 nm in a Hitachi F-2000 spectrofluorometer A buffer solution containing EP24.15 and dithiotheitol 0.5 mm (to assure maximum enzyme activation), was added to a 1-cm path-length cuvette to a final volume of mL The cuvette was placed in a thermostatically controlled cell compartment for before the substrate solution was added The increase in fluorescence was recorded continuously with time The 2989 EP24.15 membrane association and calcium sensitivity concentration of peptide solutions was obtained by colorimetric determination of the 2,4-dinitrophenyl group (17 300 m)1Ỉcm)1, extinction coefficient at 365 nm) The inner-filter effect was corrected using an empirical equation as previously described [43] All the obtained data were fitted to nonlinear least square equations, using grafit (Version 3.0, Erithacus Software Ltd, Staines, UK) Specificity rate constants (kcat ⁄ Km) were determined under first-order conditions, with substrates concentrations 10-fold less than Km The obtained first-order rate constants were divided by the total enzyme concentration to provide kcat ⁄ Km As the products Abz-GGFL, Abz-GGFLR and their respective C-terminal fragments, were resistant to hydrolysis by EP24.15, we could determine the specificity rate constants (kcat ⁄ Km) under first-order conditions, even for the peptides hydrolyzed at two peptide bonds, as previously published [3] All experiments were conducted in triplicate for at least three independent times The results varied less than 5% within these independent experiments Determination of cleaved bonds The cleaved bonds were identified by isolation of the fragments by HPLC, comparing the retention times of the products fragments with synthetic peptides encompassing the expected hydrolysis products and ⁄ or by molecular weight The molecular weights were determined by MS using a MALDI-TOF (Shimadzu Tokyo, Japan) All determinations were conducted in triplicates for at least three times independently The results varied less than 3% within these independent experiments Protein assay Protein determinations were carried out using the method described by Bradford [41] using BSA as a standard For CD experiments, protein concentration was determined according to the method described by Gill and von Hippel [43] Molecular modeling The D93 mutation was modeled onto the X-ray crystallographic structure of the recently solved human EP24.15 [17] We utilized the just released coordinates from the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics (ID 1S4B) The atomic coordinates of this structure served as a template for the D93, a conserved residue The in silico substitution of the mutated alanine, as well as model building, were calculated and rendered with the computer software suites quanta, insight ii (Accelrys, San Diego, CA, USA), or o, for molecular visualization [44] The model was energy minimized using Molecular Operating Environment software 2990 V Oliveira et al (Montreal, Canada), and the graphics rendered with spock and raster3d [45] Acknowledgements Thanks are due to Dr Ian A Smith (Baker Institute) who provided substrate and inhibitors for EP24.15 The technical support of Sandra Regina S Lascosck, Edson Rocha de Oliveira, Gaspar Ferreira de Lima and Roberto Cabado Modia Junior is gratefully acknowledged This work was supported by the Sao Paulo State ˜ Research Foundation (FAPESP; grants 96 ⁄ 05904-8, 97 ⁄ 10831-2, 99 ⁄ 01983-9, 00 ⁄ 04297-8, 01 ⁄ 07544-9 and 04 ⁄ 04933-2), CNPq and CAPES, and NIH NS39892 and RR19325 (to MJG) PAGG and CCR were supported by studentships from FAPESP (02 ⁄ 09861-4 and 97 ⁄ 05500-7, respectively) This work is part of the PhD thesis of PAGG References Camargo AC, Gomes MD, Reichl AP, Ferro ES, Jacchieri S, Hirata IY & Juliano L (1997) Structural features that make oligopeptides susceptible substrates for hydrolysis by 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of protein-dye binding Anal Biochem 72, 248–254 Sreerama N & Woody RW (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism Anal Biochem 209, 32–44 Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326 Jones TA, Zou JY, Cowan SW & Kjeldgaard A (1991) Improved methods for building protein models in electron density maps and the location of errors in these models Acta Crystallogr A 47, 110–119 Merritt EA & Bacon DJ (1997) Raster3D: photorealistic molecular graphics Methods Enzymol 277, 505–524 FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS ... investigated the calcium effects on EP24.15 secondary structure, enzymatic activity and substrate specificity Enzymatic activity of the mutated 2981 EP24.15 membrane association and calcium sensitivity... cleavage after calcium addition (data not shown) Discussion The most important finding of this report is that calcium regulates the membrane association, secondary structure and substrate specificity. .. the plasma membrane in a calcium dependent fashion Calcium was also shown to change the secondary structure by reduction of a-helical content and to change the substrate specificity of EP24.15 Together,