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

Interaction Between Catalytically Inactive Calpain and Calpastatin

Tác giả: Monica Averna, Roberto Stifanese, Roberta De Tullio, Enrico Defranchi, Franca Salamino, Edon Melloni and Sandro Pontremoli

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

Nội dung tài liệu: Nghiên cứu này khám phá sự tương tác giữa calpain không hoạt động về mặt xúc tác và calpastatin, cung cấp bằng chứng về sự tồn tại của chúng trong các tế bào được kích thích. Các thay đổi cấu trúc của phân tử calpain khi tương tác với các ligand tự nhiên, bao gồm Ca2+, cơ chất và calpastatin, đã được theo dõi bằng kháng thể đơn dòng đặc hiệu. Nghiên cứu chỉ ra rằng calpastatin là ligand hiệu quả nhất trong việc thúc đẩy sự thay đổi cấu trúc này, dẫn đến tăng ái lực liên kết với kháng thể đơn dòng. Các thí nghiệm trên tế bào nguyên vẹn cho thấy những thay đổi cấu trúc tương tự xảy ra khi tế bào được kích thích, gợi ý một vai trò mới của calpastatin trong việc kiểm soát mức độ hoạt hóa calpain thông qua sự di chuyển đến các vị trí tác động cụ thể.

Mục lục chi tiết:

• Introduction
• Keywords
• Correspondence
• (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.
• In recent years, information has accumulated on the 3D structure of μ-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 conformational 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 activation in vivo, which has not been previously possible because of the lack of reliable techniques for evaluating active calpain species and their intracellular localization.
• 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 procedure 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 catalytic domain [32], it is possible to detect conformational changes in the calpain 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
• 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 methodology, 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 calpastatin in the cytosolic compartment.
• These data suggest 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 µg 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+ concentration, is 50% of the maximal at 25 µm Ca2+ and maximal at 100 µm 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 calpain 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 tested.
• In the presence of Ca2+ concentrations ranging from zero to 5 µm (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 molecular aggregations or dissociation of the oligomers, which have been shown to occur at higher concentrations of the metal ion [45].
• The addition of E64, a synthetic 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
• tration of 5 µm did not affect the RNCAST104-mediated increase in light emission.
• Thus in contrast with what occurs in the presence of Ca2+, the addition of equimolar amounts of calpastatin 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 previous observations indicating that calpain and calpastatin 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 calpain 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 concentration of 2 mg·mL−¹ (Fig. 4).
• The observations so far reported indicate that calpain can exist in two freely convertible inactive conformations.
• 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
• 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 corresponds approximately to a 1:1 protease/inhibitor molar ratio.
• The addition of Ca2+ even at a concentration of 5 µm did not affect the RNCAST104-mediated increase in light emission.
• Thus in contrast with what occurs in the presence of Ca2+, the addition of equimolar amounts of calpastatin 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 previous observations indicating that calpain and calpastatin 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 calpain 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 concentration of 2 mg·mL−¹ (Fig. 4).
• The observations so far reported indicate that calpain can exist in two freely convertible inactive conformations.
• 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
• (physiological amounts) Ca2+ concentrations, a digestible 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, calpain undergoes conformational changes that could be detected by mAb 56.3 binding.
• For this purpose, human neutrophils were stimulated with the chemotactic 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 fluorescence 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 present in the low-affinity form (Fig. 6A).
• Scanning the fluorescence throughout the cell revealed that the protease 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 localized 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
• plasma membrane is an obligatory step in the directing of the protease to its sites of action, the preferred calpain 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 interaction of calpain with calpastatin, we stimulated Jurkat 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 unaffected [49,50].
• These findings excluded the involvement of Ca2+ in the conformational change in calpain, strongly suggesting that it is the interaction with calpastatin that is responsible for the observed effects.
• Other ligands such as digestible substrates were excluded 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 cytosol for interaction with calpain.
• This hypothesis is confirmed by the effect of arachidonate 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 conditions of unmodified Ca2+ homeostasis, clearly indicates that the ligand responsible for the changes in intracellular calpain conformation is its natural inhibitor, calpastatin.
• The new conformational state acquired by calpain in these experimental conditions may represent an intermediate, but still inactive, form which is stabilized by interaction with ligands, the most efficient being calpastatin.
• Discussion
• A major problem in understanding the physiological role of calpain is the reliability of techniques capable
• 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 proposed 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 specific mAb.
• We established that this short amino-acid sequence is confined to the catalytic domain of the protease, a region known to undergo profound conformational rearrangements [21-23], leading to expression of the catalytic activity.
• In preliminary experiments we demonstrated that the affinity of calpain for its mAb increases after exposure to various ligands.
• These changes in the conditions 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 accessible.
• In this paper we report that the exposure of calpain to micromolar physiological concentrations of Ca2+, or a digestible protein substrate, or calpastatin is followed 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 conformation, 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 appropriate effectors.