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

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

1 Laboratorio de Neurociencias, Universidade da Cidade de Sao Paulo, Brazil

2 Departamento de Biologia Celular e Desenvolvimento, Programa de Biologia Celular, Instituto de Ciencias Biomedicas, Universidade de

Sao Paulo, Brazil

3 Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil

4 Departamento de Biofısica, Universidade Federal de Sao Paulo, Brazil

5 Centro de Toxinologia Aplicada (CAT), Instituto Butantan, Brazil

6 Departamento de Farmacologia, Faculdade de Ciencias Medicas, Universidade Estadual de Campinas, Campinas, Brazil

7 Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX, USA

8 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 Comunicacao

Celular, Avenida Prof. Lineu Prestes1524

Sala 431, Sao Paulo, 05508-900, SP, Brazil

E-mail: [email protected]

Note

V. Oliveira and P.A.G. Garrido contributed

equally to this work.

(Received 9 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 5 to 17 amino

acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neuro-

tensin). The constitutive secretion of EP24.15 from glioma C6 cells was

demonstrated to be stimulated linearly by reduced concentrations of extra-

cellular 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 contribu-

tion to binding of the enzyme at the cell surface. These mutated recombin-

ant proteins behave similarly to the wild-type enzyme regarding enzymatic

activity, secondary structure, calcium sensitivity and immunoreactivity.

However, immunocytochemical analyses by confocal microscopy consis-

tently 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

The metalloendopeptidase EC 3.4.24.15 (EP24.15) rep-

resents a distinctive peptide-metabolizing enzyme with

size-selectivity for peptides ranging from 8 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 isola-

ted 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, myeli-

nated and unmyelinated axons, axon terminals, and in

both astrocytes and oligodendrocytes [6]. Recent stud-

ies have shown that EP24.15 could interact with

peptides generated by the multicatalytic complex pro-

teasome [7,8]. Thus, the high concentration of the

enzyme observed inside the cells could be somewhat

related to the degradation of catabolic products gener-

ated by the proteasome [7,9]. EP24.15 has been mainly

implicated in extracellular metabolism of neuropep-

tides [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 hor-

mone as well as calcium ionophore A23187 [13]. How-

ever, 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 neu-

ropeptides, such as b-endorphin [13]. EP24.15 secretion

from glioma C6 cells is even more unusual, as it is sti-

mulated 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. Site-

directed 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

surface, and it can bind calcium as corroborated by

molecular modeling by the EP24.15 derived X-ray dif-

fraction 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 con-

ducted: soluble EP24.15 activity was determined in the

medium and subtracted from the total activity

observed in the presence of cells, the latter correspond-

ing to cells + medium activity (Fig. 1B). Total extra-

cellular EP24.15 activity (cells + media) remains the

same with respect to the presence or absence of cal-

cium (Fig. 1B). However, there is a higher EP24.15

activity in the medium of cells incubated in calcium-

free 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 experi-

ments, 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 ¼5), from a total activity in the whole cell homogenates

of 2.9 ± 0.3 mU per 106 cells (n ¼ 5), further suggest-

ing that cells were intact after incubation in calcium-

free 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 indica-

ting 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 pres-

ence (Fig. 2B) or absence (Fig. 2C) of calcium. The

presence of Triton X100 (0.1%) during the immuno-

cytochemical 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

V. Oliveira et al. EP24.15 membrane association and calcium sensitivity

FEBS Journal 272 (2005) 2978–2992 ª 2005 FEBS 2979

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

antiserum was preadsorbed with the recombinant

EP24.15 (100 lgÆmL)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 incu-

bation of the cells in calcium-free medium strongly

affects the intensity of EP24.15 extracellular immuno-

staining (Fig. 3B). These data suggest the shedding of

EP24.15 from the plasma membrane after calcium

removal. Immunocytochemistry for electron micro-

scopy 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 site-

directed mutagenesis producing the mutants D93A,

D159A and the double mutant, D93 ⁄ 159A. The corres-

ponding recombinant proteins were expressed in bac-

teria and purified to homogeneity (Fig. 5A). Western

blot analyses show that such point mutations have no

effect on antiserum recognition (Fig. 5B). The follow-

ing experiments were performed in an attempt to corre-

late a possible effect of the above point mutations with

the calcium-dependent EP24.15 membrane association.

The homogeneously purified EP24.15 recombinant pro-

teins (wild-type or mutant) were incubated with glioma

C6 cells and the putative membrane binding was ana-

lyzed 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 extra-

cellular 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 immu-

noreactivity related to EP24.15 (Fig. 6C and D). Con-

trol experiments under these conditions show that

without the addition of EP24.15 the basal immunoreac-

tivity 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 resi-

due within the three-dimensional realm of the enzyme,

Fig. 1. Calcium-dependent EP24.15 released into the medium.

EP24.15 activity in intact cells was determined using b-lipotro-

pin(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)-carboxyl-

2-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 con-

centration. (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.

EP24.15 membrane association and calcium sensitivity V. Oliveira et al.


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 dis-

tance (2.5–3 A) for calcium. The coordinates for Fig. 7

were obtained from the crystal structure of thimet

oligopeptidase deposited at the Research Collaboratory

for Structural Bioinformatics (Protein Data Bank ID

#1S4B). The figure was drawn with the computer

modeling program spock and then subsequently ren-

dered using raster 3d [16]. To ensure that electro-

statics 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

Fig. 2. EP24.15 immunoreactivity in glioma C6 cells. EP24.15 presence in glioma C6 cells was further confirmed by immunochemical reac-

tions. (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 immunocytochem-

istry 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 min in 4% formaldehyde, pH 7.4. After three cycles of NaCl ⁄ Pi washing (15 min each) cells

were incubated for 2 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 wave-

length 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 com-

posites 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 experi-

ments performed independently and exhibiting similar results.



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

that there are no gross perturbations in expression of

the mutant proteins with respect to folding. CD spec-

tra of wild-type EP24.15 and mutants without

Fig. 4. 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 des-

cribed. (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.

Fig. 3. 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 min at 4 �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 exam-

ined 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 1 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 regu-

lar image; (2) nomarski; (3) glow scale. The immunolabeling intensity is shown in the graphic (intensity grows from green to red). No immu-

nolabeling is observed after preabsorbing the anti-EP24.15 serum with recombinant EP24.15 (100 lgÆmL)1; data not shown).



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 differ-

ences 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

(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 combina-

tion 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

0 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 sub-

strate, 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 2 mm calcium incre-

ment, the kcat ⁄Km (lm)1Æs)1) ratio of both Abz-

GGFLRRVQ-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 cal-

cium regulates the membrane association, secondary

structure and substrate specificity of the endo-oligo-

peptidase EP24.15. Site-directed mutagenesis indicated

that residue D93 plays an important role in this cal-

cium-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. More-

over, biochemical analyses also indicated that calcium

affects EP24.15 secondary structure (Fig. 8) and

AWT 93 159 93/159

4- Ca2+

+ Ca2+

3

2

1

0WT D93A D159A D93/159A

EP24.15

UA

F/m

in

93 159 93/159

78kDa

78kDa

B

C

Fig. 5. SDS ⁄ PAGE and western blot of EP24.15 recombinant pro-

teins. (A) SDS ⁄ PAGE stained with Comassie blue shows the homo-

geneity of the recombinant proteins (5 lgÆlane)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 recom-

binant proteins (0.5 lgÆlane)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 meas-

ured with the fluorogenic substrate QFS in the absence and in the

presence of 2 mM CaCl2. Note that calcium slightly increases

EP24.15 enzymatic activity in these assays.



Fig. 6. Immunocytochemical staining of glioma C6 cells after incubation with the recombinant EP24.15 proteins. Glioma C6 cells were prein-

cubated at 4 �C in the presence (A–D) or absence (E) of 2 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

4 �C for 30 min in the presence of the anti-EP24.15 serum (1 : 500). The excess of primary antiserum was removed rinsing the cells three

times in 1 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. 7. 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 mini-

mized using Molecular Operating Environment software, and the figure was generated using SPOCK.



changes the enzyme substrate specificity (Table 2).

These data suggest that calcium is an important medi-

ator 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 connect-

ive 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-hor-

mone processing enzymes [21], receptors [22], neuro-

transmitters [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 environ-

ment, as demonstrated by the presence of protease

inhibitors in the cultured medium of striatal astrocytes

that prevented Met-enkephalin generation from secre-

ted proenkephalin [25–27]. Therefore, the present

results suggest that in physiological conditions

EP24.15 would be able to associate with the extracellu-

lar 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 charac-

terization of peptide-processing enzymes such as

EP24.15 in the extracellular milieu of glial cells is of

neurobiological significance.

Table 1. Calcium effects on the secondary structure of EP24.15.

a-Helix b-Strand Turn Unordered

TBS

WT 42.4% 13.9% 14.3% 26.9%

D93A 44.3% 14.7% 12.6% 25.8%

D159A 57.5% 10.6% 11.4% 22.3%

D93 ⁄ 159A 50.3% 12.3% 12.6% 24.5%

TBS + 2.2 mM Ca2+

WT 38.7% 15.6% 14.4% 27.6%

D93A 40.2% 15.9% 13.5% 27.2%

D159A 49.1% 12.7% 12.8% 25.1%

D93 ⁄ 159A 47.1% 14.6% 12.3% 25.6%

Fig. 8. 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 pres-

ence of CaCl2 2 mM (h). Note that calcium addition reduces the a-helical content of all EP24.15s analyzed (Table 1).



In a previous study we reported that EP24.15 secre-

tion 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]. Bio-

chemical observations shown here indicated that

extracellular calcium concentrations could not change

the overall rate of peptide metabolism from intact gli-

oma C6 cells in culture (Fig. 1). These data suggest

that the overall extracellular EP24.15 enzymatic activ-

ity was not affected by altered calcium concentra-

tions. 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 experi-

ments both at the light and electron microscopy

levels. Indeed, immunocytochemistry analyses shown

here suggested that EP24.15 extracellular immuno-

reactivity 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

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 pep-

tide sequence nor any other hydrophobic domains to

mediate its interaction with biological membranes [31]

and the mechanism responsible for the enzyme mem-

brane 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 associ-

ation. 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 iden-

tified in the homologue endopeptidase EP24.16; both

D93 and D159 residues are located at the external sur-

face of the enzyme [17,33]. The D93A or D93 ⁄ 159Adouble mutations were shown to affect the calcium-

dependent 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 struc-

ture, reducing the a-helical content by 3–8%. It is well

known that calcium affects the conformational struc-

ture 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 altera-

tions 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 associ-

ation remains unknown. A possible explanation that

needs further attention is that D93 mediates the inter-

action of EP24.15 with a yet unidentified membrane

protein. Molecular modeling corroborates that the resi-

due is on the surface.

Table 2. Influence of calcium concentration on EP24.15 peptide

bond cleavage.

[Ca2+]

(mM)

Cleaved bond (%)

Abz-GGFLRRVQ-EDDnp Abz-GGFLRRDQ-EDDnp

L–Rb R–Rb

kcat ⁄ Kma

(lM)1.s)1) L–Rb R–Rb

kcat ⁄Kma

(lM)1.s)1)

0 36 64 2.0 63 37 1.3

0.5 – – – 62 38 1.3

1 – – – 61 39 1.3

2 – – – 56 44 1.3

5 31 69 1.5 52 48 1.4

10 28 72 1.2 45 55 1.3

50 21 79 0.8 26 74 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%.



A previous report demonstrated the influence of cal-

cium 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]. Fluoro-

genic 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 Abz-

GGFLRRDQ-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 struc-

tural changes induced by calcium on EP24.15 secon-

dary structure could modulate its substrate specificity,

even altering the cleavage site on specific peptides.

Moreover, the results obtained with the substrate Abz-

GGFLRRDQ-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 sub-

strate Abz-GGFLRRDQ-EDDnp containing a negat-

ive 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 concentra-

tions. Similar analyses using the substrate Abz-

GGFLRRVQ-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 pub-

lished reports [3,40], and could be of importance in the

development of new specific substrates and ⁄or inhibi-

tors for the enzyme, as the detailed architecture of the

enzyme and its interactions with substrates are deci-

phered.

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 chan-

ges 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

the EP24.15 structure, which have not yet been des-

cribed.

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 secon-

dary 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 com-

plex 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 UÆmL)1

penicillin G, and 50 lgÆmL)1 streptomycin sulfate, as previ-

ously described [12]. The cells were maintained in 12-well

plates (5.2 · 105 cellsÆcm)2 density of saturation), at 37 �Cin 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 1 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, 4 gÆL)1

d-glucose, final pH 7.4) equilibrated at 37 �C. For the cal-

cium-free experiments, cells were previously incubated for

1 h in fresh NaCl ⁄Pi (0.2 m phosphate buffer pH 7.4, con-

taining 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 cellsÆcm)2)

containing 0.5 mLÆwell)1 Henk’s media. The extent of

hydrolysis was consistently <10% during all the determi-

nations. The enzymatic reactions were terminated by addi-

tion of 5 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 min at a flow rate of 2 mLÆmin)1. The rate of the

reaction was evaluated by determining the amount of

[Met5]enkephalin formed from b-lipotropin(61–69). Absorb-

ance was monitored at a wavelength of 214 nm.

[Met5]enkephalin concentrations were 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 (QF7) [16].

To discern peptidolytic activity exclusively due to EP24.15,

the inhibitors N-(1-(R,S)-carboxyl-2-phenylethyl)-AAF-p-

amino-benzoate (CFP-AAF-pAB) and ⁄ or the dipeptide

Pro–Ile were used. All enzymatic determinations were con-

ducted 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 extra-

ct ⁄media, 13 lm QF7, 1 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 CFP-

AAF-pAB but not by Pro–Ile), able to hydrolyze 1 nmol

QF7Æmin)1ÆmL)1, at 37 �C pH 7.5, in TBS containing

1 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 1 min and filtered through a 0.2-lm filter (Mil-

lipore), prior to determination of the extracellular EP24.15

and G6PD enzyme activities. Intact C6 cells from each well

(� 5.2 · 105 cellsÆcm)2) were re-suspended and homogen-

ized in 1 mL ice cold TBS using a Potter–Elvehjem homo-

genizer (10 · 30 s at 900 r.p.m). The samples were then

centrifuged (105 000 g, 60 min) and the supernatant (cyto-

solic fraction) was used to determine both EP24.15 and

G6PD enzyme activities. G6PD enzyme activity was deter-

mined as previously described [13], in a 1 mL (final volume)

reaction mixture of 0.1 m Tris ⁄HCl pH 8.0, 0.1 mm glu-

cose-6-phosphate, 1 mm NADP+, and an amount of

sample able to alter 0.05–0.1 units of absorbance at

340 nmÆmin)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 nmÆmin)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 2 min. Dye excess was removed from the cell

supernatant after a brief centrifugation (1000 g, 5 min),

and cells were counted using a Neubauer chamber

(Hirschmann). Cells that did not incorporate the dye

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 nitrocellu-

lose 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 pre-

viously [18]. For controls, the antiserum was replaced by

serum preadsorbed with 100 lgÆmL)1 of recombinant

(EP24.15). After 2 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 anti-

rabbit IgGs (1 : 5000; GE Healthcare, Amersham, UK) for

1 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 pre-

viously incubated for 1 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 min in 4% formalde-

hyde pH 7.4. After three cycles of NaCl ⁄Pi washing

(15 min each) cells were incubated 2 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 min at 4 �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% formalde-

hyde, pH 7.4. After three cycles of 15 min wash in

NaCl ⁄Pi, anti-(rabbit Cy3-conjugated) Ig (1 : 250; Sigma)

diluted in NaCl ⁄Pi containing 0.01% Triton X100, 3% nor-

mal goat serum and 1% BSA was added to the cells, and

incubated for 2 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 lgÆmL)1) and incubation of the preimmune

serum (1 : 4000) instead of the anti-EP24.15 serum. Follow-

ing several cycles of washing in NaCl ⁄Pi, the cover slips

were mounted over slide glass using glycerol. The immuno-

fluorescent staining was observed and photographed on



either a microscope equipped for epifluorescence with the

corresponding filters (data not shown), or on a confocal

microscope as described below. Labeled cells were exam-

ined 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 charac-

teristics of the original signal.

For electron microscopy, cells were processed using a

pre-embedding procedure [6]. Briefly, cells were fixed at

4 �C for 1 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 min 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 1 min, and thawed in

phosphate buffer at room temperature. Thawed cells were

incubated for 30 min in TBS containing 3% of normal goat

serum, and then for 18 h at 4 �C in rabbit anti-EP24.15

serum diluted 1 : 1000 in TBS containing 0.5% normal goat

serum. Cells were rinsed in 0.01 m NaCl ⁄Pi (0.01 m phos-

phate buffer pH 7.4, containing 0.9% NaCl), incubated for

2 h in a 1 : 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 min in 2% glutaraldehyde in 0.01 m NaCl ⁄Pi. After

several washes in 0.2 m citrate buffer, pH 7.4, the immuno-

gold was silver-enhanced by incubating the sections for

7 min with IntenSE M silver solution (Amersham). The

reaction was stopped by washing in citrate buffer, and post-

fixed in 2% osmium tetroxide in phosphate buffer for

40 min, dehydrated in graded ethanols and flat-embedded

in Polybed 812 (Polysciences, Inc., Warrington, Pennsylva-

nia, 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. Ultra-

thin sections were counterstained with lead citrate and

uranyl acetate and examined with a JEOL 1010 TEM. Neg-

atives 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

Biotech Inc.) using the protocols described by the manufac-

tory of the Quick-change Site-directed mutagenesis kit (Strat-

agene, Inc). Oligonucleotide primers 5¢-CGTGTCTCCGAA

CAAGGCAATCCGCGCAGC-¢3 (sense) and 5¢-GCTGCG

CGGATTGCCTTGTTCGGAGACACG-3¢ (antisense) and

5¢-CCACTTACCTCAGGCAACACAGGAGAAGATCAAG-

3¢ (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 muta-

tions screened by automatic DNA sequencing, using a

MegaBace machine (GE Healthcare). Plasmid DNA contain-

ing the desired mutation was purified (Mini-Prep, Promega

Corp.) and transformed into electrocompetent DH5a bacter-

ial cells and plated overnight on plates containing ampicillin

to yield single colonies. Expression and purification of the

mutant proteins for biochemical characterization were per-

formed 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, 3 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 cm2Ædmol)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 SEL-

CON algorithm [42].

Kinetic assays

The hydrolysis of the quenched fluorescence peptide sub-

strates at 37 �C in TBS was followed by measuring the

fluorescence at kem ¼ 420 nm and kex ¼ 320 nm in a Hita-

chi 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 2 mL. The cuvette was placed

in a thermostatically controlled cell compartment for 5 min

before the substrate solution was added. The increase in

fluorescence was recorded continuously with time. The



concentration of peptide solutions was obtained by

colorimetric determination of the 2,4-dinitrophenyl group

(17 300 m)1Æcm)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 previ-

ously published [3]. All experiments were conducted in trip-

licate 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 frag-

ments 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 deter-

minations 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 crystallo-

graphic 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 coordi-

nates 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 mini-

mized using Molecular Operating Environment software

(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 Rober-

to 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 suppor-

ted by studentships from FAPESP (02 ⁄09861-4 and

97 ⁄ 05500-7, respectively). This work is part of the PhD

thesis of PAGG.

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

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