THROMBOSIS RESEARCH 66; 757-764,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
REPLACEMENT OF MEGAKARYOCYTES
m-CALPAIN BY )I-CALPAIN DURING MATURATIUN OF AND POSSIBLE INVOLVEMENT IN PLATELET FORMATION
Megumi Nakamural, Mayumi Moriz, Shinpei Nakazawa3, Tsuyoshi Tange4, Masami Hayashil, Yumiko Saitol, and Seiichi Kawashimal," Tokyo Metropolitan Institute of IDepartment of Biochemistry, =Department of Hematology, Gerontology, Tokyo Metropolitan Hospital, 3Department of Pediatrics, Geriatric School of Medicine, Keio University, and *Department of Pathology, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
(Received
13.2.1992;
accepted
in revised form 8.51992
by Editor A. Takada)
ABSTRACT Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca2+-requiring form (mcalpain) and the low-Caz'-requiring form (p-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-u-calpain, and staining with anti-p-calpain was prominent only in megakaryocytes, To confirm the result, megakaryoblastic cell line (T-33) cells were subjected to immunoblot analysis. However no immunoreactivity to p-calpain was seen in T-33 cell$. 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-p-calpain These results suggest that )1-calpain plays a antibody. crucial role in mature megakaryocytes, possibly in platelet production.
--_-______-_---_---_
calpain, localization, human bone marrow, megakaryocyte "To whom correspondence should be addressed. Present address: Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
Key words:
757
758
CALPAINS IN MEGAKARYOCYTES
Vol. 66, No. 6
INTRODUCTION Calpains (EC 3.4.22.17) and its endogenous inhibitor calpadistributed in mammalian and avian are ubiquitously statin Despite considerable knowledge of the chemical tissues (l-3). properties and catalytic mechanism of calpain, its physiological functions remain obscure. Several studies have suggested that calpain is responsible for mitosis (4) and cell fusion In an attempt to elucidate the physiological role of (5,6). calpain, we are examining the changes in calpain during the As the first step, we used immunomaturation of blood cells. histochemical techniques to study the cellular localization in bone marrow, which contains various kinds of cells at different In this report, we describe the cellular stages of maturation. in these cells with and p-calpain of m-calpain distribution special reference to the maturation of megakaryocytes. MATERIALS AND METHODS Materials--Blood and bone marrow were obtained with informed consent from hematologically normal subjects and patients with malignant lymphoma, and so on. Megakaryocyte cell line (T-33) is from the megakaryoblastic crisis of a patient suffering from chronic myelogenous leukemia. Monoclonal antibodies specific to m-calpain and p-calpain prepared by the hybridoma technique described previously The monoclonal anti-m-calpain (3AllD12) and anti-p-calpain [&A2) antibodies were specific for the large catalytic subunits of their respective antigens, and showed no cross-reaction with The clone 3E4 is any other protein on immunoblot analysis. negative against both calpains and was used as a control.
were
met&o&--1mmunohistochemical mmunobistocbemical analysis was performed essentially as described before (8). Smears of bone marrow or blood were dried and fixed in methanol at room temperature and incubated with 0.3% Hz02 in methanol for 5 min to block endogenous peroxidase activity. The samples were incubated with 20% goat serum and then reacted with the primary antibodies in 10% goat serum-0.05% Tween at 4°C overnight. After washing, the horseradish incubated with peroxidase-conjugated were slides anti-mouse Fab' fragment at room temperature for 1 h and rinsed. The reaction product was developed by incubation of the sample in 0.05% 3,3'-diaminobenzidine tetrahydrochloride-0.009% HzOz-0.05 M Tris/HCl-0.01 M imidazole, pH 7.6. The samples were then counterstained with Meyer's hematoxylin and mounted. Western blot analysis--Cells were boiled for 5 min in the sample SDS-polyacrylamide gel electrophoresis buffer and subjected to according to the procedure of Laemmli (9). Proteins were transferred from gels to nitrocellulose membranes by the method of Blotted proteins were first incubated with Towbin et al. (10). anti-p-calpain antibodies anti-m-calpain or and then with peroxidase-conjugated anti-mouse Fab' fragment. The peroxidase stainings were developed using 3,3' -diaminobenzidine tetrahydrochloride as the substrate.
Vol. 66, No. 6
759
CALPAINS IN MEGAKARYOCYTES RESULTS
Localization of m-calpain and p-calpain in human bone marrow cells--Figure 1 illustrates typical immunohistochemical staining
reactions. No cell was stained by the control antibody (Fig. m-Calpain was localized to erythroblasts, myeloid cells, la). and plasma cells (Fig. lb). The contents in erythrocytes and platelets were below the limits of detection since Western blot analysis of isolated cells showed immunoreactive bands. Thus, a lack of staining indicates a relatively low level rather than a total absence of calpains. Anti-u-calpain antibody reacted strongly only with megakaryocytes as shown in Fig. lc. Western blot analysis and imunocytochemical staining of cell line cells--To confirm the strong immunostaining of megakaryo-
cytes in bone marrow by anti-p-calpain antibody, we also examined the distribution of both calpains in the cells of an established megakaryoblastic cell line (T-33) by Western blotting and immunoSurprisingly, T-33 cells reacted only histochemical methods. with the anti-m-calpain and not with the anti-p-calpain antibody, in contrast with the immunohistochemical staining of bone marrow smears described above (Figs. 2 and 3). Figure 3e illustrates distinct staining of a smear of T-33 cells by the anti-m-calpain antibody. Again, u-calpain could not be detected in these mega-
IImmunohistochemical distribution of m-calpai,n and Fig. 1. The bone marrow smears were u-calpain in human bone marrow. stained with antibodies and then counterstained by Meyer's Therefore, in the control specimen, nuclei were hematoxylin. stained blue but no cell was immunostained. a, 3E4 (control); b,3AllD12 (for m-calpain); c, lA8A2 (for pl-calpain). M, myelocyte; Ebl, erythroblast; Mgk, megakaryocyte. Bar, 50 p.
760
CALPAINS IN MEGAKARYOCYTES
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m-Calpain is karyoblastic cells by this method (Fig. 3b). localized in very immature cells with mononuclei as shown in Fig. 3c. Even the large-sized cell with an indentation of the nucleus seen in Fig. 3e is younger than the cells from bone marrow, because T-33 cells do not have demarcation membranes (11). The discrepant results between the cells of the megakaryoblastic cell line and bone marrow megakaryocytes may therefore be attributed to maturation-dependent distribution of m-calpain and p-calpain. We next examined the relationship between cellular maturation and localization of calpain in megakaryocytes. and p-calpain in megakaryocytes at Localizatl'on of m-calpain various stages of maturation---Figure 4 illustrates the immuno-
staining of a bone marrow smear from a patient with idiopathic thrombocytopenic purpura, a disease characterized by richness in The degree of megakaryocytes at various stages of maturation. was judged morphologically. megakaryocytes maturation of Immature, small mononuclear megakaryocytes (stages I and II) were stained by the anti-m-calpain, but large-sized cells with irregularly shaped nuclei (stage III) were stained much less (Fig In contrast, mature megakaryocytes reacted strongly 4b and c). with the anti-p-calpain antibody, while only poor staining of immature cells was observed (Fig. 4d and e).
B t
113K88K t
*,,
t
t
55K -
Mab c
:.
Mabc
Fig. 2. Western blot analysis of m-calpain and u-calpain in the cells of a megakaryoblastic cell line (T-33). Cell lysate (6 x lo* cells) were subjected to SDS-PAGE on 10% polyacrylamide gels The blotted proteins (lane c) were immunostained with antibodies against m-calpain (3AllD12; panel A) and p-calpain (lA8A2; panel The m-calpain (lane a) and p-calpain (lane b) used as D). standards were purified from rabbit skeletal muscle. Molecular weights of the prestained marker proteins (lane M) are shown at the bottom in kilodaltons.
Vol. 66, No. 6
CALPAINS IN MEGAKARYOCYTES
761
Fig. 3. Localization of p-calpain and m-calpain in the cells of a Smears of cultured cells were megakaryoblastic cell line (T-33). fixed in methanol and reacted with monoclonal antibodies followed by staining with peroxidase-conjugated anti-mouse Fab' fragment. The samples were then counterstained by Meyer's hematoxylin. 3E4 (control); b, lA8A2 (for p-calpain); c, d, and e, 3AllDTi In panels a and b, only blue staining for (for m-calpain). In panels c, nuclei were seen but no cells were immunostained. d, and e, cells immunostained by anti-m-calpain were shown in Bar, 50 p_ order of maturation. DISCUSSION Calpain has been purified from blood cells such as erythroleukocytes (13), and polymorphonuclear (12), platelets and little is known about the distribution However, We physiological role of calpain in hematopoietic cell systems. first demonstrated the localization of m-calpain and p-calpain in While erythroblasts were immunostained human bone marrow cells. by the anti-m-c;::::; antibody, m-calpain was absent in erythroduring that m-calpain disappears shows The cytes. The time of disappearance should be maturation of erythrocytes. around of enucleation since nucleated chicken erythrocytes still ;;$s
762
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Fig. 4. The change in immunostaining of m-calpain and )I-calpain during maturation of megakaryocytes. Bone marrow smears from a patient with idiopathic thrombocytopenic purpura were fixed and immunostained as described in tne method section. 3E4 (for (control); b and c, 3AllD12 (for m-calpain); d and e, IA& p-calpain). The degree of maturation of the megakaryocytes was Arrowheads indicate megakaryocytes. judged morphologically. Bar, 50 p. contain m-calpain (15). Surprisingly, no immunoreactive band for anti-p-calpain antibody was detected in megakaryoblastic T-33 cells in spite of strong staining of megakaryocytes in bone Instead, m-calpain was found in the place of marrow smears. However, the p-calpain in the megakaryoblastic cell lines. immunohistochemical results from bone marrow smears from a patient with idiopathic thrombocytopenic purpura, characterized by richness in megakaryocytes at various developmental stages, offered further evidence that calpain is related to the maturation, and consequently the function of megakaryocytes. Although the mechanism of platelet formation from the cytoplasm of megakaryocytes has not been elucidated, exocytosis seems to be involved (16). Various proteases have been implicated to play a role in fusion processes such as formation of multinucleated myotubes (19). (5,17), fertilization (18) and exocytosis Recently we demonstrated that membrane proteolysis catalyzed by u-calpain is required for fusion of erythrocytes induced by the membrane-mobility agent AzC and Caz+ (20). Thus, p-Calpain is likely to play an essential role in the protein alterations
Vol. 66, No. 6
CALPAINS IN MEGAKARYOCYTES
763
required for membrane fusion. Furthermore, it is considered that cytoskeletal proteins such as vimentin and lining proteins such as spectrin are good substrates for calpain (21). This strongly suggests the involvement of p-calpain in the special function of megakaryocytes, possibly in the formation of platelets through exocytosis catalyzing the membrane fusion. ACKNOWLEDGEHJ3NT
We are grateful to Mr. Yoshihiro Fujita for taking photographs. REFERENCES 1.
KAWASHIMA,S., NOMOTO,M., HAYASHI,M., INOMATA,M., NAKAMURA, M. and IMAHORI,K. Comparison of calcium-activated neutral proteases from skeletal muscle of rabbit and chicken. J. Bz*ochem. 95, 95-101, 1984.
2. KAWASHIMA,S., HAYASHI,M., SAITO,Y., KASAI,Y. and IMAHORI,K. Tissue distribution of calcium-activated neutral proteinases in rat. Biochlqm. Biophys. Acta 965, 130-135 (1988). 3. NAKAMURA,M., IMAHORI,K. and KAWASHIMA,S. Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats. Comp. Biochem. Physl.01. 89B, 381-384, 1988. 4. SCHOLLMEYER,J.E. Calpain II involvement in mitosis. 240, 911-913 (1988)
Science
5. SCHOLLMEYER,J.E. Role of Ca2+ and Caz+-activated protease in myoblast fusion. Exp. Cell Res. 162, 411-422 (1986) 6. KOSOWER,N.S., GLASER,T. and KOSOWER,E.M. Membrane-mobility agent-promoted fusion of erythrocytes: Fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins. Proc. Natl. Acad. Sci. USA SO, 7542-7546 (1983) 7. KASAI,Y., INOMATA,M., HAYASHI,M., IMAHORI,K. and KAWASHIMA, S. Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity. J. Biochem. 100, 183-190, 1986. 8. HAWKS,R., NIDAY,E. and GORDON,J. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147, 1982. 9. LAEMMLI,U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685, 1970. 10. TOWDIN,H., STAEHLIN,T. and GORDON,J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some application. Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1979)
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11. TANGE,T., NAKAHARA,K., MITANI,K., YAMASAKI,I., YASUDA,H., TANAKA,F., MIZUGUCHI,M., ODA,H., YATOMI,Y., TAKANASHI,R., FUJIOKA,S., YAMAGUCHI,K. and URANO,Y. Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis. Cancer Res. 48, 6137-6144, 1908. 12. KITAHARA,A., SASAKI,T., KIKUCHI,T., YUMOTO,N., YOSHIMURA, N HATANAKA,M. and MURACHI,T. Large-scale purification of po.&ine calpain I and calpain II and comparison of proteolytic fragments of their subunits. J. Biochem. 95, 17591766, 1984. 13. YOSHIDA,N., WEKSLER,B. and NACHMAN,R. Purification of human platelet calcium-activated protease: Effect on platelet and endothelial function. J. Biol. Chem. 258, 7168-7174, 1983. 14. FUKUI,I., TOYOHARA,H., ITO,K., HAMAKUBO,T. and MURACHI,T. Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes. Biochemistry 27, 3260-3267, 1988. 15. MURAKAMI,T., UEDA,M., HAMAKUBO,T. and MURACHI,T. Identification of both calpains I and II in nucleated chicken erythrocytes. J. Bl.ochem. 103, 168-171, 1980. 16. WILLIAMS,W.J., BEUTLER,E., ERSLEV,A.J. and LICHTMAN,M.A. Hematology. New York: McGraw-Hill, ~~1186, 1983. 17. COUCH,C.B. and STRITTMATTER,W.J. Rat myoblast fusion requires metalloendoprotease activity. Cell 32, 257-265, 1983. 18. WATANABE,N., VANDE WOUDE,G.F., IKAWA,Y. and SAGATA,N. Specific proteolysis of the c-mosproto-oncogene product by calpain on fertilization of Xenopus eggs. Nature 342, 505511, 1989. 19. BAKTER,D.A., JOHNSTON,D. and STRITTMATTER,W.J. Protease inhibitors implicate metalloendoprotease in synaptic transmission at the mammalian neuromuscular junction. Proc. Natl. Acad. Sci. USA 80, 4174-4178, 1903. 20. HAYASHI,M., SAITO,Y. and KAWASHIMA,S. Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Caz+. B~Dochenz.Bl*ophys. Res. Comzun. 182, 939-946, 1992. 21. INOMATA,M., HAYASHI,M., NAKAMURA,M., IMAHORI,K. and KAWASHIMA,S. Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP). J. Biochem. 98, 407-416, 1985.
REPLACEMENT OF MEGAKARYOCYTES
m-CALPAIN BY )I-CALPAIN DURING MATURATIUN OF AND POSSIBLE INVOLVEMENT IN PLATELET FORMATION
Megumi Nakamural, Mayumi Moriz, Shinpei Nakazawa3, Tsuyoshi Tange4, Masami Hayashil, Yumiko Saitol, and Seiichi Kawashimal," Tokyo Metropolitan Institute of IDepartment of Biochemistry, =Department of Hematology, Gerontology, Tokyo Metropolitan Hospital, 3Department of Pediatrics, Geriatric School of Medicine, Keio University, and *Department of Pathology, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
(Received
13.2.1992;
accepted
in revised form 8.51992
by Editor A. Takada)
ABSTRACT Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca2+-requiring form (mcalpain) and the low-Caz'-requiring form (p-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-u-calpain, and staining with anti-p-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 p-calpain was seen in T-33 cell$. 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-p-calpain These results suggest that )1-calpain plays a antibody. crucial role in mature megakaryocytes, possibly in platelet production.
--_-______-_---_---_
calpain, localization, human bone marrow, megakaryocyte "To whom correspondence should be addressed. Present address: Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
Key words:
757
758
CALPAINS IN MEGAKARYOCYTES
Vol. 66, No. 6
INTRODUCTION Calpains (EC 3.4.22.17) and its endogenous inhibitor calpadistributed in mammalian and avian are ubiquitously statin Despite considerable knowledge of the chemical tissues (l-3). properties and catalytic mechanism of calpain, its physiological functions remain obscure. Several studies have suggested that calpain is responsible for mitosis (4) and cell fusion In an attempt to elucidate the physiological role of (5,6). calpain, we are examining the changes in calpain during the As the first step, we used immunomaturation of blood cells. histochemical techniques to study the cellular localization in bone marrow, which contains various kinds of cells at different In this report, we describe the cellular stages of maturation. in these cells with and p-calpain of m-calpain distribution special reference to the maturation of megakaryocytes. MATERIALS AND METHODS Materials--Blood and bone marrow were obtained with informed consent from hematologically normal subjects and patients with malignant lymphoma, and so on. Megakaryocyte cell line (T-33) is from the megakaryoblastic crisis of a patient suffering from chronic myelogenous leukemia. Monoclonal antibodies specific to m-calpain and p-calpain prepared by the hybridoma technique described previously The monoclonal anti-m-calpain (3AllD12) and anti-p-calpain [&A2) antibodies were specific for the large catalytic subunits of their respective antigens, and showed no cross-reaction with The clone 3E4 is any other protein on immunoblot analysis. negative against both calpains and was used as a control.
were
met&o&--1mmunohistochemical mmunobistocbemical analysis was performed essentially as described before (8). Smears of bone marrow or blood were dried and fixed in methanol at room temperature and incubated with 0.3% Hz02 in methanol for 5 min to block endogenous peroxidase activity. The samples were incubated with 20% goat serum and then reacted with the primary antibodies in 10% goat serum-0.05% Tween at 4°C overnight. After washing, the horseradish incubated with peroxidase-conjugated were slides anti-mouse Fab' fragment at room temperature for 1 h and rinsed. The reaction product was developed by incubation of the sample in 0.05% 3,3'-diaminobenzidine tetrahydrochloride-0.009% HzOz-0.05 M Tris/HCl-0.01 M imidazole, pH 7.6. The samples were then counterstained with Meyer's hematoxylin and mounted. Western blot analysis--Cells were boiled for 5 min in the sample SDS-polyacrylamide gel electrophoresis buffer and subjected to according to the procedure of Laemmli (9). Proteins were transferred from gels to nitrocellulose membranes by the method of Blotted proteins were first incubated with Towbin et al. (10). anti-p-calpain antibodies anti-m-calpain or and then with peroxidase-conjugated anti-mouse Fab' fragment. The peroxidase stainings were developed using 3,3' -diaminobenzidine tetrahydrochloride as the substrate.
Vol. 66, No. 6
759
CALPAINS IN MEGAKARYOCYTES RESULTS
Localization of m-calpain and p-calpain in human bone marrow cells--Figure 1 illustrates typical immunohistochemical staining
reactions. No cell was stained by the control antibody (Fig. m-Calpain was localized to erythroblasts, myeloid cells, la). and plasma cells (Fig. lb). The contents in erythrocytes and platelets were below the limits of detection since Western blot analysis of isolated cells showed immunoreactive bands. Thus, a lack of staining indicates a relatively low level rather than a total absence of calpains. Anti-u-calpain antibody reacted strongly only with megakaryocytes as shown in Fig. lc. Western blot analysis and imunocytochemical staining of cell line cells--To confirm the strong immunostaining of megakaryo-
cytes in bone marrow by anti-p-calpain antibody, we also examined the distribution of both calpains in the cells of an established megakaryoblastic cell line (T-33) by Western blotting and immunoSurprisingly, T-33 cells reacted only histochemical methods. with the anti-m-calpain and not with the anti-p-calpain antibody, in contrast with the immunohistochemical staining of bone marrow smears described above (Figs. 2 and 3). Figure 3e illustrates distinct staining of a smear of T-33 cells by the anti-m-calpain antibody. Again, u-calpain could not be detected in these mega-
IImmunohistochemical distribution of m-calpai,n and Fig. 1. The bone marrow smears were u-calpain in human bone marrow. stained with antibodies and then counterstained by Meyer's Therefore, in the control specimen, nuclei were hematoxylin. stained blue but no cell was immunostained. a, 3E4 (control); b,3AllD12 (for m-calpain); c, lA8A2 (for pl-calpain). M, myelocyte; Ebl, erythroblast; Mgk, megakaryocyte. Bar, 50 p.
760
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m-Calpain is karyoblastic cells by this method (Fig. 3b). localized in very immature cells with mononuclei as shown in Fig. 3c. Even the large-sized cell with an indentation of the nucleus seen in Fig. 3e is younger than the cells from bone marrow, because T-33 cells do not have demarcation membranes (11). The discrepant results between the cells of the megakaryoblastic cell line and bone marrow megakaryocytes may therefore be attributed to maturation-dependent distribution of m-calpain and p-calpain. We next examined the relationship between cellular maturation and localization of calpain in megakaryocytes. and p-calpain in megakaryocytes at Localizatl'on of m-calpain various stages of maturation---Figure 4 illustrates the immuno-
staining of a bone marrow smear from a patient with idiopathic thrombocytopenic purpura, a disease characterized by richness in The degree of megakaryocytes at various stages of maturation. was judged morphologically. megakaryocytes maturation of Immature, small mononuclear megakaryocytes (stages I and II) were stained by the anti-m-calpain, but large-sized cells with irregularly shaped nuclei (stage III) were stained much less (Fig In contrast, mature megakaryocytes reacted strongly 4b and c). with the anti-p-calpain antibody, while only poor staining of immature cells was observed (Fig. 4d and e).
B t
113K88K t
*,,
t
t
55K -
Mab c
:.
Mabc
Fig. 2. Western blot analysis of m-calpain and u-calpain in the cells of a megakaryoblastic cell line (T-33). Cell lysate (6 x lo* cells) were subjected to SDS-PAGE on 10% polyacrylamide gels The blotted proteins (lane c) were immunostained with antibodies against m-calpain (3AllD12; panel A) and p-calpain (lA8A2; panel The m-calpain (lane a) and p-calpain (lane b) used as D). standards were purified from rabbit skeletal muscle. Molecular weights of the prestained marker proteins (lane M) are shown at the bottom in kilodaltons.
Vol. 66, No. 6
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761
Fig. 3. Localization of p-calpain and m-calpain in the cells of a Smears of cultured cells were megakaryoblastic cell line (T-33). fixed in methanol and reacted with monoclonal antibodies followed by staining with peroxidase-conjugated anti-mouse Fab' fragment. The samples were then counterstained by Meyer's hematoxylin. 3E4 (control); b, lA8A2 (for p-calpain); c, d, and e, 3AllDTi In panels a and b, only blue staining for (for m-calpain). In panels c, nuclei were seen but no cells were immunostained. d, and e, cells immunostained by anti-m-calpain were shown in Bar, 50 p_ order of maturation. DISCUSSION Calpain has been purified from blood cells such as erythroleukocytes (13), and polymorphonuclear (12), platelets and little is known about the distribution However, We physiological role of calpain in hematopoietic cell systems. first demonstrated the localization of m-calpain and p-calpain in While erythroblasts were immunostained human bone marrow cells. by the anti-m-c;::::; antibody, m-calpain was absent in erythroduring that m-calpain disappears shows The cytes. The time of disappearance should be maturation of erythrocytes. around of enucleation since nucleated chicken erythrocytes still ;;$s
762
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Fig. 4. The change in immunostaining of m-calpain and )I-calpain during maturation of megakaryocytes. Bone marrow smears from a patient with idiopathic thrombocytopenic purpura were fixed and immunostained as described in tne method section. 3E4 (for (control); b and c, 3AllD12 (for m-calpain); d and e, IA& p-calpain). The degree of maturation of the megakaryocytes was Arrowheads indicate megakaryocytes. judged morphologically. Bar, 50 p. contain m-calpain (15). Surprisingly, no immunoreactive band for anti-p-calpain antibody was detected in megakaryoblastic T-33 cells in spite of strong staining of megakaryocytes in bone Instead, m-calpain was found in the place of marrow smears. However, the p-calpain in the megakaryoblastic cell lines. immunohistochemical results from bone marrow smears from a patient with idiopathic thrombocytopenic purpura, characterized by richness in megakaryocytes at various developmental stages, offered further evidence that calpain is related to the maturation, and consequently the function of megakaryocytes. Although the mechanism of platelet formation from the cytoplasm of megakaryocytes has not been elucidated, exocytosis seems to be involved (16). Various proteases have been implicated to play a role in fusion processes such as formation of multinucleated myotubes (19). (5,17), fertilization (18) and exocytosis Recently we demonstrated that membrane proteolysis catalyzed by u-calpain is required for fusion of erythrocytes induced by the membrane-mobility agent AzC and Caz+ (20). Thus, p-Calpain is likely to play an essential role in the protein alterations
Vol. 66, No. 6
CALPAINS IN MEGAKARYOCYTES
763
required for membrane fusion. Furthermore, it is considered that cytoskeletal proteins such as vimentin and lining proteins such as spectrin are good substrates for calpain (21). This strongly suggests the involvement of p-calpain in the special function of megakaryocytes, possibly in the formation of platelets through exocytosis catalyzing the membrane fusion. ACKNOWLEDGEHJ3NT
We are grateful to Mr. Yoshihiro Fujita for taking photographs. REFERENCES 1.
KAWASHIMA,S., NOMOTO,M., HAYASHI,M., INOMATA,M., NAKAMURA, M. and IMAHORI,K. Comparison of calcium-activated neutral proteases from skeletal muscle of rabbit and chicken. J. Bz*ochem. 95, 95-101, 1984.
2. KAWASHIMA,S., HAYASHI,M., SAITO,Y., KASAI,Y. and IMAHORI,K. Tissue distribution of calcium-activated neutral proteinases in rat. Biochlqm. Biophys. Acta 965, 130-135 (1988). 3. NAKAMURA,M., IMAHORI,K. and KAWASHIMA,S. Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats. Comp. Biochem. Physl.01. 89B, 381-384, 1988. 4. SCHOLLMEYER,J.E. Calpain II involvement in mitosis. 240, 911-913 (1988)
Science
5. SCHOLLMEYER,J.E. Role of Ca2+ and Caz+-activated protease in myoblast fusion. Exp. Cell Res. 162, 411-422 (1986) 6. KOSOWER,N.S., GLASER,T. and KOSOWER,E.M. Membrane-mobility agent-promoted fusion of erythrocytes: Fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins. Proc. Natl. Acad. Sci. USA SO, 7542-7546 (1983) 7. KASAI,Y., INOMATA,M., HAYASHI,M., IMAHORI,K. and KAWASHIMA, S. Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity. J. Biochem. 100, 183-190, 1986. 8. HAWKS,R., NIDAY,E. and GORDON,J. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147, 1982. 9. LAEMMLI,U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685, 1970. 10. TOWDIN,H., STAEHLIN,T. and GORDON,J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some application. Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1979)
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CALPAINS IN MEGAKARYOCYTES
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11. TANGE,T., NAKAHARA,K., MITANI,K., YAMASAKI,I., YASUDA,H., TANAKA,F., MIZUGUCHI,M., ODA,H., YATOMI,Y., TAKANASHI,R., FUJIOKA,S., YAMAGUCHI,K. and URANO,Y. Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis. Cancer Res. 48, 6137-6144, 1908. 12. KITAHARA,A., SASAKI,T., KIKUCHI,T., YUMOTO,N., YOSHIMURA, N HATANAKA,M. and MURACHI,T. Large-scale purification of po.&ine calpain I and calpain II and comparison of proteolytic fragments of their subunits. J. Biochem. 95, 17591766, 1984. 13. YOSHIDA,N., WEKSLER,B. and NACHMAN,R. Purification of human platelet calcium-activated protease: Effect on platelet and endothelial function. J. Biol. Chem. 258, 7168-7174, 1983. 14. FUKUI,I., TOYOHARA,H., ITO,K., HAMAKUBO,T. and MURACHI,T. Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes. Biochemistry 27, 3260-3267, 1988. 15. MURAKAMI,T., UEDA,M., HAMAKUBO,T. and MURACHI,T. Identification of both calpains I and II in nucleated chicken erythrocytes. J. Bl.ochem. 103, 168-171, 1980. 16. WILLIAMS,W.J., BEUTLER,E., ERSLEV,A.J. and LICHTMAN,M.A. Hematology. New York: McGraw-Hill, ~~1186, 1983. 17. COUCH,C.B. and STRITTMATTER,W.J. Rat myoblast fusion requires metalloendoprotease activity. Cell 32, 257-265, 1983. 18. WATANABE,N., VANDE WOUDE,G.F., IKAWA,Y. and SAGATA,N. Specific proteolysis of the c-mosproto-oncogene product by calpain on fertilization of Xenopus eggs. Nature 342, 505511, 1989. 19. BAKTER,D.A., JOHNSTON,D. and STRITTMATTER,W.J. Protease inhibitors implicate metalloendoprotease in synaptic transmission at the mammalian neuromuscular junction. Proc. Natl. Acad. Sci. USA 80, 4174-4178, 1903. 20. HAYASHI,M., SAITO,Y. and KAWASHIMA,S. Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Caz+. B~Dochenz.Bl*ophys. Res. Comzun. 182, 939-946, 1992. 21. INOMATA,M., HAYASHI,M., NAKAMURA,M., IMAHORI,K. and KAWASHIMA,S. Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP). J. Biochem. 98, 407-416, 1985.
