Báo cáo khoa học: Interaction between catalytically inactive calpain and calpastatin Evidence for its occurrence in stimulated cells docx

Two conformational states of calpain can be distinguished on the basis of their affinity for this mAb: the native state shows low affinity, whereas binding of specific ligands induces Keywo

Evidence for its occurrence in stimulated cells

Monica Averna, Roberto Stifanese, Roberta De Tullio, Enrico Defranchi, Franca Salamino,

Edon Melloni and Sandro Pontremoli

Department of Experimental Medicine (DIMES), Section of Biochemistry and Centre of Excellence for Biomedical Research (CEBR), University of Genova, Italy

In recent years, information has accumulated on the

3D structure of l-calpain and m-calpain [1–10], as well

as their isolated catalytic cores [11–16] Much less is

known about the process by which calpain is activated

[3,4,6,17,18] It is generally accepted that it is initiated

by the binding of calcium to several sites localized in

both calpain subunits and completed by a

conforma-tional change in domain II [19–29] However, the role

of the two Ca2+-binding sites recently identified in this

catalytic domain is still to be defined [23] More

intriguing is the possibility of detecting calpain

activa-tion in vivo, which has not been previously possible

because of the lack of reliable techniques for

evaluat-ing active calpain species and their intracellular

local-ization

Identification of autolyzed calpain forms by means

of a specific monoclonal antibody does not seem to be

of a general use, as recent structural acquisitions have

suggested that calpain activation can also proceed through a reversible process [1,2,4] The proposed pro-cedure involving the identification of calpain-degraded target proteins appears not to be sufficiently specific because of the very large number of calpain substrates present in the cell (for reviews see [3,4,9,30]) The recently devised fluorescence resonance energy transfer technology has greatly improved the sensitivity, but not the selectivity, required for the precise evaluation

of calpain activation and activity [31]

In this paper we report that, by means of a specific monoclonal antibody that recognizes the calpain cata-lytic domain [32], it is possible to detect

conformation-al changes in the cconformation-alpain molecule that occur after it binds to its natural effectors Two conformational states of calpain can be distinguished on the basis of their affinity for this mAb: the native state shows low affinity, whereas binding of specific ligands induces

Keywords

activation; calcium; calpain; calpastatin;

conformational states

Correspondence

S Pontremoli, DIMES-Section of

Biochemistry, Viale Benedetto XV,

1–16132 Genova, Italy

Fax: +39 010518 343

Tel: +39 010353 8128

E-mail: pontremoli@unige.it

(Received 9 January 2006, accepted 15

February 2006)

doi:10.1111/j.1742-4658.2006.05180.x

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease None of these conformational states showed catalytic activity and probably repre-sent intermediate forms preceding the active enzyme state In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca2+, substrate or calpasta-tin, the affinity increases up to 10-fold, with calpastatin being the most effective This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca2+] or extensive diffusion of calpast-atin into the cytosol without affecting Ca2+homeostasis The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain–calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action

transition to a conformation with significantly higher

affinity The most extensive conformational change is

induced by calpastatin; the addition of substrate or

Ca2+ proved to be less effective Using this

methodo-logy, we have shown similar molecular transitions in

calpain in intact cells stimulated with agents known to

induce either a limited increase in intracellular [Ca2+]

or extensive redistribution and accumulation of

cal-pastatin in the cytosolic compartment These data

sug-gest a new role for calpastatin in controlling the extent

of calpain translocation to and activation at specific

sites of action

Results

To study the interaction of calpain with its natural

effectors, the purified protease isolated from human

erythrocytes was immobilized on a nitrocellulose

sheet and detected by a specific mAb that recognizes

the DI-DII polypeptide in both native calpain and the

fragment that accumulates during trypsin digestion

[14,24] (Fig 1A) In Fig 1B we provide evidence that

calpain bound to nitrocellulose reacts with mAb 56.3,

generating a light signal the intensity of which is a

function of the amount of mAb used, with a saturation

value at a concentration equal to 0.5–0.75 lg antibody

After exposure of the immobilized calpain to a mixture

of Ca2+ and a digestible substrate, catalytic activity

can be detected, demonstrating that immobilization on

the nitrocellulose sheet does not modify its catalytic

properties This is demonstrated by the data in

Fig 1C, which indicate that the catalytic activity of

immobilized calpain, as a function of Ca2+

concentra-tion, is 50% of the maximal at 25 lm Ca2+ and

maximal at 100 lm Ca2+, as occurs when soluble

native enzyme is used [3,4,9,20,30] Furthermore,

inhibition of the immobilized enzyme by E64 or

calpastatin was retained (Fig 1D) The efficiency of

both inhibitors was identical with that observed in a

control assay using soluble enzyme (data not shown)

Together these results indicate that immobilized

cal-pain is an appropriate tool for the study of the effects

of natural ligands in changing its conformation, and

that these can be monitored by evaluating the intensity

of the light signal generated by the binding of mAb

56.3

To perform these investigations, the two preferential

ligands of calpain, Ca2+ions and calpastatin, were

tes-ted In the presence of Ca2+ concentrations ranging

from zero to 5 lm (close to physiological values), a

twofold increase in the intensity of the signal was

detected, in the absence of any appreciable proteolytic

activity (Fig 2) The concentrations of Ca2+ used in

these experiments were selected to avoid undesired and confusing changes in calpain structure such as mole-cular aggregations or dissociation of the oligomers, which have been shown to occur at higher concentra-tions of the metal ion [45] The addition of E64, a syn-thetic inhibitor of calpain, did not modify the intensity

of the signal observed in the presence of Ca2+ alone The increase in the mAb-binding capacity of calpain observed in these conditions can be ascribed to increased accessibility of the calpain epitope recognized

by this mAb As this conformational transition does not lead to the expression of catalytic activity, these structural changes must precede the calpain active state

As shown in Fig 3, when calpain was exposed to increasing concentrations of recombinant calpastatin RNCAST104, there was a much greater increase in the

Fig 1 Properties of nitrocellulose-immobilized native human eryth-rocyte calpain (A) Human erytheryth-rocyte calpain (10 lg) was

incubat-ed in the absence (lane 1) or presence of trypsin in a calpain ⁄ trypsin ratio of 1000 : 1 (lane 2) Western blot analysis was performed [41] using mAb 56.3 (B) Immobilized calpain was incu-bated in 0.1 mL 50 m M sodium borate buffer, pH 7.5, containing

10 m M EDTA and the indicated amounts of mAb 56.3 The immu-noreactive spots (see inset) were quantified with a Shimadzu CS9000 densitometer and expressed as arbitrary units (C) Immobil-ized calpain (0.5 lg) was assayed as described in [33] using human denatured globin as substrate for 60 min at 37
C in the presence

of the indicated [Ca2+] The nitrocellulose sheet was removed before the addition of trichloroacetic acid (7% final concentration) Calpain activity was quantified after the release of free NH2groups

as in [33] (D) Activity of immobilized calpain was assayed as in (C)

in the presence of 1 m M Ca2+and 10 l M E64 or 25 nmol rat brain recombinant calpastatin RNCAST104.

light signal (7–8-fold) than when calpain was exposed

to Ca2+alone (Fig 2) Moreover, the maximum effect

was reached with 10 nmol RNCAST104, which

corres-ponds approximately to a 1 : 1 protease⁄ inhibitor

molar ratio The addition of Ca2+ even at a

concen-tration of 5 lm did not affect the RNCAST104-medi-ated increase in light emission

Thus in contrast with what occurs in the presence of

Ca2+, the addition of equimolar amounts of calpasta-tin induces an increase of approximately one order of magnitude in the affinity of calpain for mAb 56.3, indicating that, in these conditions, the mAb epitope

on the protease has become more accessible

These data not only indicate that calpastatin induces

a pronounced Ca2+-independent change in calpain conformation, but also provide strong support for pre-vious observations indicating that calpain and calpast-atin can associate in a 1 : 1 molar ratio, regardless of the presence of Ca2+(unpublished work)

In addition to Ca2+ and calpastatin, a number of digestible substrates can behave as calpain ligands and may accordingly induce changes in the conformation

of the protease detectable by the mAb binding To investigate this, we exposed immobilized calpain to digestible and nondigestible proteins and evaluated their efficiency in promoting conformational change in the protease BSA, a protein not digested by calpain, had no effect at any concentration tested Casein, a cal-pain substrate [4,9,30], induced a progressive increase

in the binding of mAb 56.3 to calpain, as revealed by

a 2.5–3-fold increase in the light signal at a concentra-tion of 2 mgÆmL)1(Fig 4)

The observations so far reported indicate that cal-pain can exist in two freely convertible inactive confor-mations The former is mostly present in the absence

of any effector, and the other is induced, with different degrees of efficiency, by interaction with micromolar

Fig 2 Effect of Ca2+ on binding of mAb 56.3 to

nitrocellulose-immobilized calpain Immobilized calpain was incubated in 0.1 mL

50 m M sodium borate buffer, pH 7.5, in the presence of 10 m M

EDTA or the indicated Ca2+ concentration (d) After saturation,

mAb 56.3 was added and the binding of the mAb to calpain was

measured as described in Experimental procedures and the legend

to Fig 1B and expressed as arbitrary units Alternatively,

immobi-lized calpain was exposed to Ca2+ in the presence of 10 l M E64

(s) Immobilized calpain was also incubated in 0.1 mL 50 m M

sodium borate buffer, pH 7.5, containing the indicated Ca 2+

concen-tration in the presence of human denatured globin as substrate,

and its activity was measured as described in the legend to Fig 1C

(n) The results are expressed as the arithmetical mean ± SD from

four different experiments.

Fig 3 Effect of calpastatin on the binding of mAb 56.3 to

immobil-ized calpain Immobilimmobil-ized calpain was exposed to increasing

con-centrations of recombinant rat brain calpastatin RNCAST104 in the

presence of 10 m M EDTA (h) or 5 l M Ca 2+ (d) The binding of

mAb 56.3 to immobilized calpain was measured as described in

Figs 1 and 2 The data obtained in these experiments were also

analyzed by Scatchard plot (inset), using the relationship [B] ⁄ [F] ¼

k(B max – B), where [B] is the bound ligand concentration, [F] is the

free ligand concentration, B max is the maximum ligand amount, and

k is the affinity constant.

Fig 4 Effect of calpain digestible or nondigestible proteins on the binding of mAb 56.3 to immobilized calpain Immobilized calpain was mixed with 10 m M EDTA (control) in the presence of 10 pmol rat brain recombinant calpastatin RNCAST104 (black bar) or the indic-ated amounts of BSA (light grey bars) or casein (heavy grey bars) The mAb bound to immobilized calpain was detected as described

in Experimental procedures and quantified as reported in the legend to Fig 2.

(physiological amounts) Ca2+concentrations, a

digest-ible protein substrate, and finally calpastatin

The finding that calpastatin was the most efficient

ligand at promoting calpain transition, detected as an

increase in mAb that bound to calpain, is consistent

with the fact that the protease has an affinity for its

protein inhibitor that is more than 10 000-fold higher

than that for its substrates

When native calpain was replaced with the autolyzed

75-kDa form, identical results were obtained in all the

experimental conditions tested (data not shown) This

indicates that the removal of part of the DI and DV

domain from the calpain molecule does not abolish the

conformational transition described above and may

explain the Ca2+dependence of the autolyzed enzyme

[34]

We then explored whether, in stimulated cells,

cal-pain undergoes conformational changes that could be

detected by mAb 56.3 binding For this purpose,

human neutrophils were stimulated with the

chemotac-tic peptide f-Met-Leu-Phe, which is known to promote

intracellular mobilization of Ca2+ [46–48] As shown

in Fig 5A,B and quantified in Fig 5C, under these

conditions, an approximately 10-fold increase in fluor-escence emission was detected, indicating that calpain had undergone a transition from the low to the high affinity mAb-binding form Interestingly, these results obtained in vivo were almost superimposable on those obtained in vitro after exposure of native calpain to calpastatin This suggests that the same process is operating in both experimental situations

To establish if the data for human neutrophils could

be reproduced in a different cell line, we used murine erythroleukemia (MEL) cells stimulated with the Ca2+ ionophore A23187 In resting cells, calpain was poorly stained by the mAb, indicating that it was mainly pre-sent in the low-affinity form (Fig 6A) Scanning the fluorescence throughout the cell revealed that the pro-tease is quite homogeneously diffuse throughout the cytosol After stimulation with the Ca2+ ionophore (Fig 6B), the intensity of calpain staining increased 7– 8-fold, indicating that the protease was now mainly present in the high-affinity form

In these conditions, although the highest amount of calpain is still in the cytosol, a small fraction is locali-zed at the membrane, as indicated by the two small fluorescent peaks detectable at both sides of the cell scan This further confirms that translocation to the

A

C

B

Fig 5 Binding of mAb 56.3 to calpain in human neutrophils

stimul-ated with f-Met-Leu-Phe Purified neutrophils (10 7 cells) were

incub-ated at 37
C for 10 min in 10 m M Hepes, pH 7.5, (10 mL),

containing 140 m M NaCl, 5 m M MgCl 2 , 5 m M glucose and 50 l M

Ca 2+ in the absence (A) or presence (B) of 1 l M f-Met-Leu-Phe.

Cells were fixed and permeabilized as described in Experimental

procedures They were then exposed to mAb 56.3, and its binding

to calpain was detected by confocal microscopy, after incubation

with fluorescein-labeled secondary antibody (C) Fluorescence was

measured as described in Experimental procedures The data

repre-sent the arithmetical mean ± SD of four different experiments.

A

B

Fig 6 Binding of mAb 56.3 to calpain in MEL cells loaded with

Ca 2+ MEL cells (10 7 cells) were incubated at 37
C for 10 min in

10 m M Hepes, pH 7.5 (10 mL), containing 140 m M NaCl, 5 m M

MgCl 2 , 5 m M glucose and 50 l M Ca2+in the absence (A) or pres-ence (B) of 1 l M A23187 Ca 2+ ionophore Cells were fixed and per-meabilized as described in Experimental procedures They were then exposed to mAb 56.3, and its binding to calpain was detected

by confocal microscopy, after incubation with fluorescein-labeled secondary antibody The fluorescence detected in each section (0.5 lm) is shown at the right of each picture The arrow points to the calpain ring around the cell.

plasma membrane is an obligatory step in the directing

of the protease to its sites of action, the preferred

cal-pain substrates being transmembrane or

membrane-associated proteins [3,4,30]

We still required direct evidence of the nature of the

ligand responsible for the observed conformational

transition Thus, to discriminate between the effect due

to the rise in free Ca2+from that induced by the

inter-action of calpain with calpastatin, we stimulated

Jur-kat cells with arachidonate, which is known to induce

apoptosis without producing, during the early phase

of stimulation, appreciable changes in intracellular

free [Ca2+] [49,50] As previously observed (Fig 7B)

in these cells, stimulation with ionophore A23187

promoted a calpain-mediated fluorescence increase of

7–8-fold However, a sixfold increase in fluorescence

intensity was observed after brief stimulation with

arachidonate, indicating that a similar conformational

transition of calpain can be obtained in conditions in

which intracellular Ca2+ homeostasis is almost

unaf-fected [49,50] These findings excluded the involvement

of Ca2+ in the conformational change in calpain,

strongly suggesting that it is the interaction with

cal-pastatin that is responsible for the observed effects

Other ligands such as digestible substrates were exclu-ded a priori because they would never be present in the cytosol at suitable concentrations

The different calpain fluorescence observed in control (Fig 7A) and arachidonate-stimulated (Fig 7C) cells after detection with the calpain mAb can be ascribed to the presence of large amounts of calpastatin which, after stimulation, becomes freely available in the cyto-sol for interaction with calpain

This hypothesis is confirmed by the effect of arachi-donate treatment on the intracellular distribution of calpastatin (Fig 8) In untreated cells, cytosol contains

a very limited amount of calpastatin, the bulk of the inhibitor being localized in perinuclear aggregates After stimulation with arachidonate, the cell image is completely reversed, as calpastatin becomes freely diffuse in the cytosol and only traces of aggregates remain located in the perinuclear region Thus, the increased availability of calpastatin, occurring in con-ditions of unmodified Ca2+ homeostasis, clearly indi-cates that the ligand responsible for the changes in intracellular calpain conformation is its natural inhib-itor, calpastatin

The new conformational state acquired by calpain in these experimental conditions may represent an interme-diate, but still inactive, form which is stabilized by inter-action with ligands, the most efficient being calpastatin

Discussion

A major problem in understanding the physiological role of calpain is the reliability of techniques capable

Fig 7 Binding of mAb 56.3 to calpain in Jurkat cells loaded with

Ca2+or stimulated with arachidonate Jurkat cells (107cells) were

incubated at 37
C for 10 min in 10 m M Hepes, pH 7.5 (10 mL),

containing 140 m M NaCl, 5 m M MgCl 2 , 5 m M glucose and 50 l M

Ca2+in the absence (A) or presence of (B) 1 l M A23187Ca2+

-iono-phore or (C) 100 l M arachidonate Cells were fixed and

permeabil-ized as described in Experimental procedures They were then

exposed to mAb 56.3, and its binding to calpain was detected by

confocal microscopy, after incubation with fluorescein-labeled

sec-ondary antibody The data represent the arithmetical mean ± SD of

four different experiments The arrow points to the calpain ring

around the cell.

Fig 8 Effect of arachidonate on the intracellular distribution of cal-pastatin in Jurkat cells Jurkat cells were incubated with arachido-nate as described in the legend to Fig 7C After 30 min of incubation, cells were fixed, and calpastatin was probed with

7 lgÆmL)1mAb 35.23 [35] followed by a fluorescein-labeled second-ary antibody Fluorescence was quantified using the software as described in Experimental procedures Cell nuclei were stained with propidium iodide The arrows indicate the perinuclear calpasta-tin aggregates C ¼ control; Ar ¼ arachidonate.

of detecting the changes in its conformation that

accompany its activation and regulation in specific cell

compartments In spite of several attempts to solve this

problem [19–29], no precise information is available,

because of the inadequacy of the methods so far

pro-posed for evaluating the interaction of calpain with

natural ligands, a process that must occur in defined

intracellular compartments and presumably precedes

activation of the protease We explored the possibility

of approaching this problem by taking advantage of

the different accessibility of a calpain epitope to a

spe-cific mAb We established that this short amino-acid

sequence is confined to the catalytic domain of the

protease, a region known to undergo profound

con-formational rearrangements [21–23], leading to

expres-sion of the catalytic activity

In preliminary experiments we demonstrated that

the affinity of calpain for its mAb increases after

expo-sure to various ligands These changes in the

condi-tions preserving the native conformation of the

protease were interpreted as the result of a molecular

transition converting the native form into a state in

which the mAb epitope sequence becomes more

acces-sible

In this paper we report that the exposure of calpain

to micromolar physiological concentrations of Ca2+,

or a digestible protein substrate, or calpastatin is

fol-lowed by a change in its conformation, which can be

monitored by an increase in its affinity for its mAb

Calpain does not show catalytic activity in any of these

conditions, indicating that this molecular transition

precedes the onset of the active enzyme form Using a

Scatchard plot as a calibration curve, we established

that calpastatin promotes the conversion of almost all

the calpain molecules into the high-affinity

conforma-tion, whereas the other ligands promote the transition

of only 20–30% of the calpain molecules Thus, this

procedure provides a tool for the identification of the

calpain states generated by its interaction with natural

ligands

This methodology was successfully applied to intact

cells, and the results show that similar conformational

changes in calpain occur after stimulation with

appro-priate effectors

In human neutrophils and MEL cells, even though

the limited increase in Ca2+ could be regarded as the

event that promoted these changes, two relevant

find-ings suggest a different conclusion The first concerns

the extent of the increase in calpain fluorescence, which

could not be induced by Ca2+alone as indicated in the

in vitro experiments (Figs 2 and 3) The second is

rela-ted to the availability of calpastatin in the cytosol, the

concentration of which increases when Ca2+

homeosta-sis is perturbed, as previously reported [43] Thus, the pronounced change in calpain conformation revealed

by the high fluorescence reached can only be attributed

to the interaction of calpain with calpastatin

Stimulation of Jurkat cells with arachidonate provi-ded experimental evidence in favor of this hypothesis

as it simultaneously promoted mobilization of calpast-atin accompanied by a marked transition in the cal-pain molecule, suggesting its interaction with the inhibitor Moreover, these events occur in the early phase of Jurkat cell stimulation, in which no evidence for alteration in Ca2+ homeostasis has been obtained [49,50] Thus, the effect of arachidonate suggests that conditions promoting calpain–calpastatin interaction in the cytosol can be Ca2+ independent and precede cal-pain activation Additional information was obtained using this methodology in experiments with MEL cells stimulated with a Ca2+ionophore Analysis of the dis-tribution of calpain in the cells revealed that, in addi-tion to the massive conformaaddi-tional change in the enzyme present in the cytosol, an increase in intracellu-lar [Ca2+] also induces translocation of a small amount of the protease to the plasma membrane These results are in agreement with the accepted evi-dence that an increase in intracellular [Ca2+] induces degradation of some proteins specifically localized at the inner surface of the plasma membrane [3,4,30] and with the observation that the extent of calpain activa-tion at the plasma membrane is a funcactiva-tion of the amount of cytosolic calpastatin [43]

Taken together, these findings suggest that the for-mation of a calpain–calpastatin complex before the onset of calpain activation is functionally relevant, not only for the modulation of calpain activation in the cytosol, but also for controlling the amount of calpain translocated to and activated at the plasma membrane

Experimental procedures

Materials

Ca2+ionophore A23187, f-Met-Leu-Phe, arachidonic acid, BSA, casein, nonfat skimmed milk powder, trypsin, and E64 were purchased from Sigma Aldrich (Milan, Italy)

Purification of human erythrocyte calpain and recombinant rat brain calpastatin

Human erythrocyte calpain was purified and assayed as reported in [33] Autoproteolyzed human erythrocyte cal-pain (75 kDa) was prepared as described in [34] Rat brain recombinant calpastatin RNCAST104 (GenBank accession number Y13588) was prepared as indicated in [35]

Monoclonal antibodies

The mAb against calpain (mAb 56.3) was obtained as

reported in [32] It showed inhibitory properties when

added to a calpain activity assay mixture [32,36] The

mAb against calpastatin (mAb 35.23) was produced as

described in [35]

Cell culture and human neutrophil isolation

MEL cells were obtained and cultured as specified in [37]

Jurkat cells (human T-lymphocyte line) were cultured at

37
C (5% CO2) in RPMI 1640 (Sigma Aldrich) growth

medium containing 10% fetal bovine serum (Euroclone

Ltd, UK), 10 UÆmL)1 penicillin (Sigma Aldrich),

100 lgÆmL)1streptomycin (Sigma Aldrich) and 4 mm

l-glu-tamine Human neutrophil isolation was based on a

modifi-cation [38] of the procedure described in [39]

Calpain digestion by trypsin

Human erythrocyte calpain (10 lg) was incubated in 50 lL

50 mm sodium borate buffer, pH 7.5, containing 0.5 mm

2-mercaptoethanol and 1 mm EDTA for 60 min at room

tem-perature with or without trypsin in a calpain⁄ trypsin ratio of

1000 : 1 [14] After 60 min, 50 lL 120 mm Tris⁄ HCl,

pH 6.8, containing 4% SDS, 4% 2-mercaptoethanol and

20% glycerol was added to the incubation mixture and then

heated for 3 min at 100
C Samples (50 lL) were submitted

to SDS⁄ PAGE (10% gel) [40], and western blot was

per-formed as indicated in [41] Proteins were probed with mAb

56.3 The immunoreactive material was revealed as reported

in [42]

Calpain immobilization

The procedure for immobilization of human erythrocyte

calpain is summarized in Scheme 1 The purified enzyme

(0.5 lg in 5 lL 50 mm sodium borate buffer, pH 7.5, con-taining 0.1 mm EDTA) was spotted on a nitrocellulose sheet (0.5 cm· 0.5 cm; Bio-Rad Laboratories, Bio-Rad Ita-lia, Milan, Italy) and left for 15 min at 4
C in a humidified chamber The sheet was then washed with 1 mm EDTA and saturated with 5% nonfat skimmed milk powder The nitrocellulose sheet was then incubated in 0.1 mL sodium borate buffer, pH 7.5, containing the calpain ligands in the conditions specified elsewhere The mixtures were incubated

at 4
C for 30 min mAb 56.3 (0.2 lg) was then added Calpain was detected using a peroxidase-conjugated secon-dary antibody [42] developed with an ECL detection sys-tem (Amersham Pharmacia Biotech)

Immunoreactive material was detected by subjecting the probed nitrocellulose sheets to autoradiography and quanti-fied with a Shimadzu CS9000 densitometer using a fixed wavelength of 590 nm

Binding of mAb 56.3 to intracellular calpain revealed by confocal microscopy and fluorescence quantification

Control or stimulated MEL cells, Jurkat cells or human neu-trophils were washed with NaCl⁄ Pi Calpain and calpastatin were detected by confocal analysis as described in [43], using mAb 56.3 or mAb 35.23 respectively as primary antibody and a fluorescein isothiocyanate-conjugated sheep anti-mouse IgG as secondary antibody (Amersham Biosciences Europe Gmbh, Milan, Italy) The excitation⁄ emission wave-lengths were 488⁄ 522 nm for fluorescein-labeled antibodies and 488–568⁄ 605 nm for propidium iodide-stained chroma-tin [44] Fluorescence was quantified with Laser Pix Software (Bio-Rad Bioscience)

Acknowledgements

This work was supported in part by grants from MIUR, FIRB and PRIN projects, and from the

Uni-Scheme 1.

versity of Genova We thank Mr Roberto Minafra for

his skilful technical assistance

References

1 Moldoveanu T, Campbell RL, Cuerrier D & Davies PL

(2004) Crystal structures of calpain–E64 and –leupeptin

inhibitor complexes reveal mobile loops gating the

act-ive site J Mol Biol 343, 1313–1326

2 Hosfield CM, Elce JS & Jia Z (2004) Activation of

cal-pain by Ca2+: roles of the large subunit N-terminal and

domain III–IV linker peptides J Mol Biol 343, 1049–

1053

3 Pontremoli S, Melloni E & Salamino F (1999)

Cal-pain: from structure to biological function In Calcium

as a Cellular Regulation (Carafoli E & Klee C, eds),

pp 371–388 Oxford University Press, New York

4 Goll DE, Thompson VF, LIH, Wei W & Cong J (2003)

The calpain system Physiol Rev 83, 731–801

5 Pal GP, De Veyra T, Elce JS & Jia Z (2003)

Purificat-ion, crystallization and preliminary X-ray analysis of a

mu-like calpain Acta Crystallogr D Biol Crystallogr 59,

369–371

6 Maki M, Kitaura Y, Satoh H, Ohkouchi S & Shibata H

(2002) Structures, functions and molecular evolution

of the penta-EF-hand Ca2+-binding proteins Biochim

Biophys Acta 1600, 51–60

7 Reverter D, Braun M, Fernandez-Catalan C, Strobl S,

Sorimachi H & Bode W (2002) Flexibility analysis

and structure comparison of two crystal forms of

cal-cium-free human m-calpain Biol Chem 383, 1415–

1422

8 Dainese E, Minafra R, Sabatucci A, Vachette P,

Mello-ni E & CozzaMello-ni I (2002) Conformational changes of

cal-pain from human erythrocytes in the presence of Ca2+

J Biol Chem 277, 40296–40301

9 Sorimachi H & Suzuki K (2001) The structure of

cal-pain J Biochem (Tokyo) 129, 653–664

10 Masumoto H, Nakagawa K, Iria S, Sorimachi H,

Suzuki K, Bourenkov GP, Bartunik H,

Fernandez-Cata-lan C, Bode W & Strobl S (2000) Crystallization and

preliminary X-ray analysis of recombinant full-length

human m-calpain Acta Crystallogr D Biol Crystallogr

56, 73–75

11 Pal G, De Veyra T, Elce JS & Jia Z (2003) Crystal

structure of a micro-like calpain reveals a partially

acti-vated conformation with low Ca2+requirement

Struc-ture (Camb) 11, 1521–1526

12 Leinala EK, Arthur JS, Grochulski P, Davies PL, Elce

JS & Jia Z (2003) A second binding site revealed by

C-terminal truncation of calpain small subunit, a

penta-EF-hand protein Proteins 53, 649–655

13 Wang C, Barry JK, Min Z, Tordsen G, Rao AG &

Olsen OA (2003) The calpain domain of the maize

DEK1 protein contains the conserved catalytic triad

and functions as a cysteine proteinase J Biol Chem 278, 34467–34474

14 Thompson VF, Lawson KR, Barlow J & Goll DE (2003) Digestion of mu- and m-calpain by trypsin and chymotrypsin Biochim Biophys Acta 1648, 140–153

15 Brandenburg K, Harris F, Dennison S, Seydel U & Phoenix D (2002) Domain V of m-calpain shows the potential to form an oblique-orientated alpha-helix, which may modulate the enzyme’s activity via interac-tions with anionic lipid Eur J Biochem 269, 5414– 5422

16 Cygler M, Grochulski P & Blauchard H (2002) Crystal-lization and structural details of Ca2+-induced conform-ational changes in the EF-hand domain VI of calpain Methods Mol Biol 172, 243–260

17 Moldoveanu T, Hosfield CM, Lim D, Jia Z & Davies

PL (2003) Calpain silencing by a reversible intrinsic mechanism Nat Struct Biol 10, 371–378

18 Nakagawa K, Masumoto H, Sorimachi H & Suzuki K (2001) Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and

is not required for activation J Biochem (Tokyo) 130, 605–611

19 Moldoveanu T, Jia Z & Davies PL (2004) Calpain acti-vation by cooperative Ca2+binding at two non-EF-hand sites J Biol Chem 279, 6106–6114

20 Dutt P, Spriggs CN, Davies PL, Jia Z & Elce JS (2002) Origins of the difference in Ca2+ requirement for activation of mu- and m-calpain Biochem J 367, 263–269

21 Moldoveanu T, Hosfield CM D.Lim Elce JS & Davies

PL (2002) A Ca2+switch aligns the active site of cal-pain Cell 108, 649–660

22 Jia Z, Hosfield CM, Davies PL & Elce JS (2002) Crystal structure of calpain and insights into Ca2+-dependent activation Methods Mol Biol 172, 51–67

23 Hata S, Sorimachi H, Nakagawa K, Maeda T, Abe K

& Suzuki K (2001) Domain II of m-calpain is a

Ca2+-dependent cysteine protease FEBS Lett 501, 111–114

24 Michetti M, Salamino F, Minafra R, Melloni E & Pon-tremoli S (1997) Calcium-binding properties of human erythrocyte calpain Biochem J 325, 721–726

25 Tompa P, Emori Y, Sorimachi H, Suzuki K & Friedrich

P (2001) Domain III of calpain is a Ca2+-regulated phospholipid-binding domain Biochem Biophys Res Commun 280, 1333–1339

26 Hosfield CM, Moldoveanu T, Davies PL, Elce JS & Jia

Z (2001) Calpain mutants with increased Ca2+ sensitiv-ity and implications for the role of the C(2)-like domain

J Biol Chem 276, 7404–7407

27 Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bourenkow G, Bartunik H, et al (2000) The crystal structure of calcium-free human m-calpain suggests an

electrostatic switch mechanism for activation by

cal-cium Proc Natl Acad Sci USA 97, 588–592

28 Hosfield CM, Elce JS, Davies PL & Jia Z (1999) Crystal

structure of calpain reveals the structural basis for

Ca2+-dependent protease activity and a novel mode of

enzyme activation EMBO J 18, 6880–6889

29 Blanchard H, Grochulski P, Li Y, Arthur JS, Davies

PL, Elce JS & Cygler M (1997) Structure of a calpain

Ca2+-binding domain reveals a novel EF-hand and

Ca2+-induced conformational changes Nat Struct Biol

4, 532–538

30 Croall DE & De Martino GN (1991) Calcium-activated

neutral protease (calpain) system: structure, function,

and regulation Physiol Rev 71, 813–847

31 Stockolm D, Bartoli M, Sillon G, Bourg N, Davoust J

& Richard I (2005) Imaging calpain protease activity by

multiphoton FRET in living mice J Mol Biol 346, 215–

222

32 Pontremoli S, Melloni E, Damiani G, Salamino F,

Sparatore B, Michetti M & Horecker BL (1988) Effects

of a monoclonal anti-calpain antibody on responses of

stimulated human neutrophils Evidence for a role for

proteolytically modified protein kinase C J Biol Chem

263, 1915–1919

33 Melloni E, Sparatore B, Salamino F, Michetti M &

Pontremoli S (1982) Cytosolic calcium-dependent

protei-nase of human erythrocytes: formation of an

enzyme-natural inhibitor complex induced by Ca2+ions

Biochem Biophys Res Commun 106, 731–740

34 Michetti M, Salamino F, Tedesco I, Averna M, Minafra

R, Melloni E & Pontremoli S (1996) Autolysis of

human erythrocyte calpain produces two active enzyme

forms with different cell localization FEBS Lett 392,

11–15

35 Melloni E, De Tullio R, Averna M, Tedesco I,

Salami-no F, Sparatore B & Pontremoli S (1998) Properties of

calpastatin forms in rat brain FEBS Lett 431, 55–58

36 Pontremoli S, Sparatore B, Salamino F, De Tullio R,

Pontremoli R & Melloni E (1988) The role of calpain in

the selective increased phosphorylation of the

anion-transport protein in red cell of hypertensive subjects

Biochem Biophys Res Commun 151, 590–597

37 Melloni E, Pontremoli S, Damiani G, Viotti PL, Weich

N, Rifkind RA & Marks PA (1988) Vincristine-resistant

erythroleukemia cell line has marked increased

sensitiv-ity to hexamethylene bisacetamide-induced

differentia-tion Proc Natl Acad Sci USA 85, 3835–3839

38 De Tullio R, Stifanese R, Salamino F, Pontremoli S &

Melloni E (2003) Characterization of a new p94-like

cal-pain form in human lymphocytes Biochem J 375, 689– 696

39 Boyum A (1968) Isolation of mononuclear cells and gra-nulocytes from human blood Scan J Clin Lab Invest

21, 77–89

40 Laemmli U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685

41 Towbin J, Staehelin T & Gordon J (1979) Electrophore-tic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci USA 76, 4350–4354

42 Palejwala S & Goldsmith LT (1992) Ovarian expression

of cellular Ki-ras p21 varies with physiological status Proc Natl Acad Sci USA 89, 4202–4206

43 DeTullio R, Passalacqua M, Averna M, Salamino F, Melloni E & Pontremoli S (1999) Changes in intracellu-lar localization of calpastatin during calpain activation Biochem J 343, 467–472

44 Coligan SE, Knisbeck AM, Margulies DH, Shevach

EM & Strober W (1991) Current Protocols in Immunol-ogy.Wiley, New York

45 Pal GP, Elce JS & Jia Z (2001) Dissociation and aggre-gation of calpain in the presence of calcium J Biol Chem 276, 47233–47238

46 Anderson R, Steel HS & Tintinger GR (2005) Inositol 1,4,5-triphosphate-mediated shuttling between intracellu-lar stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils Biochem Pharmacol 69, 1567–1575

47 Thompson NT, Bonser RW, Tateson JE, Spacey GD, Randall RW, Hodson HF et al (1991) A quantitative investigation into the dependence of Ca2+mobilisation

on changes in inositol 1,4,5-triphosphate levels in the stimulated neutrophil Br J Pharmacol 103, 1592–1596

48 Steel HC & Anderson R (2002) Dissociation of the PAF-receptor from NADPH oxidase and adenylate cyclase in human neutrophils results in accelerated influx and delayed clearance of cytosolic calcium Br J Pharmacol 136, 81–89

49 Doroshenko N & Doroshenko P (2004) Ca2+influx is not involved in acute cytotoxicity of arachidonic acid Biochem Pharmacol 67, 903–909

50 Farooqui AA, Ong WY & Horrocks LA (2004) Bio-chemical aspects of neurodegeneration in human brain: involvement of neural membrane phospholipids and phospholipases A2 Neurochem Res 29, 1961–1977