Localization Of Calpain Research Articles

Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma-enriched fraction, cAMP, or NGF showed concentration-dependent inhibition of both mu calpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated mu calpain (37%) and mcalpain (58%) of tSC while PDGF-aa and PDGF-bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF-aa, and PDGF-bb compared to control while this activity was 40% with FGF-treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain-calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Calpain localization changes in coordination with actin-related cytoskeleton changes during early embryonic development of Drosophila.

Calpain, a calcium-dependent intracellular protease, was identified in Drosophila melanogaster. Drosophila calpain has an amino acid sequence highly homologous to those of mammalian calpains and exhibits a distinct domain structure consisting of cysteine protease and calcium-binding domains. Specific antibodies raised against a recombinant calpain fragment were used to identify the localization of calpain in developing Drosophila embryos. Calpain was first detected near the anterior pole and in posterior region of the embryo just after fertilization. The anterior calpain disappeared during the cleavage cycles. On the other hand, the posterior calpain moved to the posterior pole when polar buds were formed, and condensed just below the pole cells. At cleavage cycles 8 and 9, when nuclei reached the egg surface, calpain was localized between the nuclei at the surface beneath the precleavage furrows. Co-staining experiments with anti-actin antibody revealed that calpain condenses specifically at the edge of and between actin caps that underlie the plasma membrane immediately above each nucleus. These results indicate that calpain is involved in the dynamic changes in the embryonic cytoskeleton, especially actin-related structures, during early embryogenesis prior to cellularization.

Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila.

Calpain, a calcium-dependent intracellular protease, was identified in Drosophila melanogaster. Drosophila calpain has an amino acid sequence highly homologous to those of mammalian calpains and exhibits a distinct domain structure consisting of cysteine protease and calcium-binding domains. Specific antibodies raised against a recombinant calpain fragment were used to identify the localization of calpain in developing Drosophila embryos. Calpain was first detected near the anterior pole and in posterior region of the embryo just after fertilization. The anterior calpain disappeared during the cleavage cycles. On the other hand, the posterior calpain moved to the posterior pole when polar buds were formed, and condensed just below the pole cells. At cleavage cycles 8 and 9, when nuclei reached the egg surface, calpain was localized between the nuclei at the surface beneath the precleavage furrows. Co-staining experiments with anti-actin antibody revealed that calpain condenses specifically at the edge of and between actin caps that underlie the plasma membrane immediately above each nucleus. These results indicate that calpain is involved in the dynamic changes in the embryonic cytoskeleton, especially actin-related structures, during early embryogenesis prior to cellularization.

Characterization of calpain II in the retina and photoreceptor outer segments

Calpain II was purified to apparent homogeneity from bovine neural retinas. It was found to be biochemically similar to brain calpain II, purified by the same procedure, with respect to: subunit mobility in SDS-polyacrylamide gel electrophoresis; Ca2+ sensitivity; inhibition by calpeptin and other cysteine protease inhibitors; and optimal pH. Semithin cryosections were immuno-labeled with antibodies specific for the catalytic subunit of calpain II. Calpain II was detected in most layers of the retina, with the most pronounced label present in the plexiform layers (synaptic regions) and the photoreceptor outer segments. In dark-adapted retinas, the label was distributed throughout the outer segments. In light-adapted retinas, outer segment labeling was concentrated in the connecting cilium, and the inner segments were labeled. A partially pure preparation of calpain II from isolated rod outer segments was found to have the same biochemical characteristics as calpain II prepared in the same way from the whole retina. The enzyme was distributed fairly evenly between the cytosolic and cytoskeletal fractions of isolated rod outer segments. Immunoblots of the rod outer segment cytoskeleton were used to determine the susceptibility of known components of the actin-based cytoskeleton to proteolysis by calpain II in vitro. Actin was not proteolyzed at all, alpha-actinin was only slowly degraded, but myosin II heavy chain was rapidly proteolyzed. Actin filaments have been shown previously to be associated with myosin II and alpha-actinin in a small domain within the connecting cilium, where they play an essential role in the morphogenesis of new disk membranes. The localization of calpain II in the connecting cilium after light exposure, combined with the in vitro proteolysis of myosin II, suggests that calpain II could be involved in light-dependent regulation of disk membrane morphogenesis by proteolysis of myosin II.

Immunohistochemical localization of calpains and calpastatin in the rabbit eye

The localization of the two Ca-activated extralysosomal proteases m-calpain and μ-calpain in the eye of the adult rabbit was examined by immunohistochemistry, using poly- and monoclonal antibodies against the corresponding rabbit antigens. Immunoreactivity against the two forms of calpains was observed in the epithelial cells on the external and internal surfaces of the cornea as well as in the epithelial cells covering the iris and ciliary body. The sclera and choroid layers showed a relatively weak immunoreactivity. Using anti m-calpain antibodies, the pigment epithelium in the retina was heavily labelled as well as the outer and inner plaxiform layers. The outer and inner borders of the Müller cells were clearly labelled. The outer segments on the receptor cells showed a strong immunorectivity for both μ-calpain and m-calpain. Lebelling was also observed in the retinal ganglion cells in tin the nerve fiber layer. The immunohistochemical localization of calpastatin, an endogenous inhibitor of both m- and μ-calpain was also examined. A high level of calpains, especially immunoreactivity was observed in the outer segments of the cells. The results may be compatible with a role for calpains, especially m-calpain, in the secretory/phagocytic process and as modulators of the cytoskeleton in cell processes.

Replacement of m-calpain by μ-calpain during maturation of megakaryocytes and possible involvement in platelet formation

Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca 2+-requiring form (m-calpain) and the low-Ca 2+-requiring form (μ-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-μ-calpain, and staining with anti-μ-calpain was prominent only in megakaryocytes. To confirm the result, megakaryoblastic cell line (T-33) cells were subjected to immunoblot analysis. However no immunoreactivity to μ-calpain was seen in T-33 cells. Bone marrow from a patient with idiopathic thrombocytopenic purpura showed immature megakaryocytes (stage II) strongly stained by anti-m-calpain antibody while mature cells (stage III) were strongly stained by anti-μ-calpain antibody. These results suggest that μ-calpain plays a crucial role in mature megakaryocytes, possibly in platelet production.

Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes

Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83 % of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changes as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83 % to 47 % of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.

Immunohistochemical and biochemical demonstration of calcium‐dependent cysteine proteinase (calpain) in calcifying cartilage of rats

Calpain is a Ca2(+)-dependent cysteine proteinase that has neutral pH optima. There are two classes of calpains that differ in their optimal calcium ion concentration for enzymatic activity. Calpain I requires a low concentration of Ca2+ for activation, and calpain II requires a much higher Ca2+ concentration. This report describes the immunohistochemical and biochemical demonstration of calpain II in calcifying cartilage in rats and also the degradation of the cartilage proteoglycan subunit by calpain II. Immunoperoxidase (peroxidase-antiperoxidase) staining of the frozen sections of the knee joint from 3-day-old and 6-day-old Wistar rats, using polyclonal antibodies against the respective heavy subunits of calpains I and II, showed positive staining only with the anti-calpain II antibody in the hypertrophic chondrocytes and surrounding cartilaginous matrix of the growth cartilage. Diethylaminoethyl-cellulose chromatography of the cartilaginous extract from 3-day-old rats showed a peak of caseinolytic activity attributable to calpain as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anti-calpain II antibody of the calpain peak demonstrated identity with the heavy subunit of calpain II (80 kDa). Proteoglycan-degrading activity of calpain was assessed using porcine kidney calpain II and the porcine articular cartilage proteoglycan subunit. After incubation in the presence of Ca2+, degradation of proteoglycan was demonstrated by the change of the elution position on Sepharose-2B chromatography. It is possible that calpain functions as one of the proteoglycan-degrading proteolytic enzymes of growth cartilage. Intracellular localization of calpain in hypertrophic chondrocytes also suggests a role in the hypertrophic process of the chondrocyte in growth cartilage.

Immunogold Electron-Microscopic Localization of Calpain I in Human Erythrocytes

With a view to understanding the function of calpain I (EC 3.4.22.17) in vivo, the localization of the enzyme was studied by immunoelectron microscopy in human erythrocytes. Thin sections of the cells embedded in Spurr's resin were exposed to solutions of monoclonal anti-calpain I or control antibodies, biotin antimouse IgG antibodies, and streptavidin-gold. Most of the calpain I (93%) was found to be distributed throughout the cytoplasm, and only 7% of the gold particles were associated with the erythrocyte membrane. Erythrocytes were Ca2+-loaded by means of the calcium ionophore A23187, and the rise in intracellular [Ca2+] was demonstrated both by crenation of the cells, and by activation of calpain which was detected by immunoblotting. The proportions of cytosolic and membrane-bound gold labelling were, however, not altered by Ca2+-loading. These results are not consistent with the hypothesis that activation of calpain requires membrane-binding.

The ultrastructural localization of calcium-activated protease "calpain" in rat brain.

Calpain I, a calcium-activated neutral protease which degrades a number of cytoskeletal proteins, has been implicated in the rapid turnover of structural proteins that may participate in synaptic plasticity. In the present study, an antibody raised against purified erythrocyte calpain I was biochemically characterized and demonstrated to specifically bind the Mr = 80,000 subunit of both rat erythrocyte and brain calpain I. This antibody was used to examine the cellular distribution of calpain I at the electron microscopic level in rat brain and spinal cord using the avidin-biotin immunocytochemical technique. Reaction product was observed throughout neuronal perikarya, within both axonal and dendritic processes, and within spine heads and necks. Postsynaptic densities in both shaft and spine synapses were also immunoreactive. Glial cell bodies and processes were densely stained. In both neurons and glia, the reaction product was deposited along cytoskeletal elements. The localization of calpain I immunoreactivity to glial processes suggests this degradative enzyme may play a role in the glial hypertrophy and process retraction seen in brain. The presence of the enzyme in spines and postsynaptic densities is consistent with the hypothesis that it is involved in the turnover of synaptic cytoskeleton, thus providing a means through which transient physiological events effect lasting changes in the chemistry and morphology of spines.

Biochemical Demonstration and Immunohistochemical Localization of Calpain in Human Skin

The biochemical properties and immunohistochemical localization of calpain, a Ca++-dependent, intracellular, nonlysosomal cysteine proteinase was examined in human skin. Human epidermal calpain I was fractionated on a DEAE-cellulose column and was found to be half-maximally activated at 3.5 microM free Ca++ and fully activated at 10 microM Ca++ as measured by casein hydrolysis. Immunoelectrophoretic blotting of calpain revealed only a single band of Mr 83,000, when the blot was made with affinity-purified anti-calpain I heavy subunit IgG. Immunohistochemical staining of normal human epidermis showed that calpain I was localized in the cytoplasm of keratinocytes in the mid to upper epidermis but not in the basal cells. In untreated psoriatic epidermis, the deposition of this proteinase was visualized weakly just beneath the stratum corneum. However, remarkable staining was observed after photochemotherapy of topical psoralen plus long-wave UV irradiation. Whether the photochemotherapy induced a quantitative increase in the amount of calpain or merely made calpain more stainable by altering the membrane remains unknown.

Selective localization of calpain I (the low-Ca 2+-requiring form of Ca 2+-dependent cysteine proteinase) in B-cells of human pancreatic islets

An immunohistochemical study was performed to localize two distinct Ca 2+-proteases (low-Ca 2+-requiring calpain I and high-Ca 2+-requiring calpain II) and their specific inhibitor (calpastatin) in human pancreas using the respective monospecific antibodies. Strongly positive staining by anti-calpain I antibody was found in pancreatic islets, specifically in B-cells, whereas the exocrine pancreatic tissue showed essentially no positive immunostaining. No such specific staining was found with anti-calpain II antibodies or anti-calpastatin antibodies. The results suggest that the Ca 2+-dependent proteolysis in B-cells can be triggered by a small rise of the intracellular Ca 2+ concentration without serious interference by the endogenous inhibitor.