Vol. 182, No. 2, 1992 January 31, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 939-946
CALPAIN ACTIVATION IS ESSENTIAL FOR MEMBRANE FUSION OF ERYTHROCYTES IN THE PRESENCE OF EXOGENOUS CA2+ Masami
Hayashi,
Yumiko
Saito
and Seiichi
Kawashima
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173, Japan Received December 20, 1991
Summary: The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis8-(2-octylcyclopropyl)octanoate (AK) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Caz+. Under these conditions, the high sensitivity form of calciumprotease (p-calpain) in activated neutral erythrocytes is p-Calpain is activated in accordance activated autolytically. that is, both erythrocyte fusion and autolytic with fusion; activation of u-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A& and Caz+. When erythrocytes are preincubated with the Caz+ ionophore A23187, both fusion and autoA leupeptin analogue, Cbz-Leulytic activation start earlier. Leu-Leu-aldehyde (ZLLLal)! inhibits both the autolytic activation of p-calpain and fusion induced by A;?C and Caz+. These results indicate that treatment of erythrocytes with AzC and Caz+, results in first an influx of Caz+ into the cells, followed by autolytic activation of u-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes. 0 1992 ncademic
Press,
Inc.
Calcium-activated neutral protease (calpain) is an intracellular thiol proteinase that is activated by calcium ions and inhibited by a specific endogenous inhibitor (calpastatin) (1,2). In most cases, there are two forms of calpain, namely, low calcium ion (PM concentrations)-requiring p-calpain and high calcium ion (mM concentrations)-requiring m-calpain. Although the primary structures of the two forms of calpain have been determined from their cDNA sequences (3), their role in cells remains unclear. . . Abbrevlatlons: SDS, sodium dodecyl sulfate; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of SDS; ZLLLal, benzyloxycarbonyl-leucyl-leucyl-leucine aldehyde; A2C, 2-(methoxyethoxy) ethyl-cis-8-(2-octylcyclopropyl)octanoate; kDa, kilo-daltons.
939
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the activation process appears to From many experiments in vitro, include autolysis in the presence of Caz+ during which the N-terminal portions of the large and small subunits are removed (1). Because the intracellular Caz+ concentration is very low, it is process is also not certain whether this activation in vl.tro Accordingly, we chose erythrocytes as model realized in viva. cells since they are the simplest cells and contain only ucalpain (4). In a previous study (5), we reported that erythrocytes undergo a change in shape from a biconcave discocytes to echinocytes and spherocytes upon treatment with ionophore A23187 and Caz+. At the same time, intracellular p-calpain is autolyzed. both the autolytic activation of u-calpain and the Furthermore, shape change induced by A23187-CaZ+ treatment are inhibited by a leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde cell-permeable (ZLLLal), that inhibits the caseinolytic activity of calpain in vl*tro very strongly. Recently, Kosower et al. reported that the fusion of erythrocytes promoted by reagents such as A2C results from an attack by calpain on membrane proteins (6). The fusion of erythrocytes could provide a very useful system in which to study the function of p-calpain in cells. In the present paper, we describe the relationship between erythrocyte fusion and the autolytic activation of p-calpain and the simultaneous inhibition of fusion and calpain activation by ZLLLal. MATERIALS AND METHODS Ionophore A23187 and AzC were purchased from Sigma Co. The protease inhibitor ZLLLal was synthesized to the method of Ito et al. (7). Erythrocytes: Heparinized blood from rats, rabbits, and healthy humans was centrifuged. Plasma and buffy coat were removed and the erythrocytes were washed three times with phosphate-buffered saline (Dulbecco's). Fusion: Erythrocytes were resuspended in buffer (pH 5.6) containing sodium acetate (75 mM), NaCl (70 mM), CaClz (1 mM) and Dextran T-70 (100 mg/ml; Pharmacia) to a hematocrit of 8 - 10% and mixed with an equal volume of AzC suspension (0.5 pi/ml) in NaCl (150 mM) prepared by sonication for 30 set (8). The cell suspensions were incubated at 37'C. Aliquots were removed at intervals for direct observation by phase-contrast microscopy or boiled in an equal volume of sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (9). Analyses of proteins and autolysis of p-calpain: After SDSPAGE, proteins were transferred from the gels to a nitrocellulose membrane. The blotted proteins were incubated with monoclonal anti-calpain antibodies and calpain was detected using horseradish peroxidase-conjugated anti-mouse IgG and diaminobenzidine. A monoclonal antibody, lAsAz, against the large subunit of rabbit u-calpain was used for rabbit and human erythrocytes, while which recognizes the large subunits of both p-calpain and lD=oA,, m-calpain from all animal species, was used for rat erythrocytes Materials:
Chemical according
940
Vol.
182,
No.
2, 1992
because only high affinity
BIOCHEMICAL
AND
1D10A7 among our with rat u-calpain
BIOPHYSICAL
monoclonal (10,ll).
RESEARCH
antibodies
COMMUNICATIONS
reacts
with
RESULT Fmdbl*lity
of
erythrocytes
and autolytic activation al. (12), fusion in rat
ofp-calpain:
As reported by Kosower et erythrocytes is induced by A2C in the presence of Caz+ at 37'C within 30 min. We have previously found that p-calpain is activated autolytically when rabbit erythrocytes are treated with the ionophore A23187 and Ca2+ (5). As shown in Fig.l., the autolytic activation of u-calpain is induced at 30 min only when rat erythrocytes are treated with both A2C and Caz+. The molecular weight of the large subunit of rat p-calpain is 77 kDa, slightly smaller than rabbit or human which are 79 kDa (13). u-calpains, The autolysis of the rat u-calpain large subunit produces a 75 kDa species directly, whereas, the autolytic conversion of the large subunit of rabbit p-calpain is from 79 kDa to 76 kDa via a 77 kDa intermediate. We examined the specificity of our monoclonal antibodies and found that the large subunits of rabbit and human p-calpain are very similar in their reactivities. Since the monoclonal antibody
Ca2+
-+-+
A2C
-
113K
w
88~
e
fusion
-+-+ -
+
+
-
-
+
+
-1 -
-
12
-
-
+
3 4
-
-
587
-
77K 75K
+
8
Fiq. 1. Autolytic activation of intracellular p-calpain in rat erythrocytes and fusion by treatment with A,C and/or Ca2+. Erythrocytes were incubated in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of 0.5 mM CaCl= and in the presence (lanes 3, 4, 7, and 8) or absence (lanes 1, 2, 5, and 6) of 0.25 pi/ml AK. The reaction was started by incqbation at 37'C and stopped after 30 min (lanes l-4) or 60 min (lanes 5-8) by the addition of an equal volume of SDS-sample buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blotted proteins were incubated with an anti-calpain monoclonal antibody, 1D,,A7, and visualized using peroxidase-conjugated anti-mouse IgG and diaminobenzidine. 941
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
raised against the rabbit u-calpain nizes rat p-calpain only slightly, reacts with both u- and m-calpains was used for rat u-calpain. When in the presence of A2C and Ca2+ At this point, started to lyse. continued and the large subunit of and was no longer MW-products antibody. Differences
in
rates
fUSl*On
of
RESEARCH COMMUNICATIONS
large subunit (1AeA2) recoga monoclonal antibody that in all animal species (1DIOA7) rat erythrocytes were incubated for 60 min, the erythrocytes the autolysis of p-calpain u-calpain was degraded to lowrecognized by the monoclonal
erythrocytes
frOn2
Varl*OUS
species:
The time required for fusion in the presence of A2C and Ca2+ varies depending on the animal species. While rat erythrocytes start to fuse after 30 min, rabbit erythrocytes require 150 min and human erythrocytes 240 min (Fig.3). for fusion (Fig.Z), Autolytic activation of p-calpain starts at the same time as fusion (Figs.2 and 3). Effect of A.23187 on A.&-induced fusl*on: In previous studies we have found that p-calpain is activated and degrades cytoskeletal proteins when Ca I+-influx is increased by the addition of the Caz+ ionophore A23187 (5). In order to establish whether Caz+-influx affects the fusion process, we added A23187 to the fusion system. Human erythrocytes were preincubated with A23187 and fusion was started by the addition of A2C and CaZ+. In this case, autolytic activation began earlier than in the absence of A23187, but was
Ca*+
0 0.5
A2C
--+
fusion
_
0 0.25 0.5 ++
-
12345
-
-
-
0 0.5
0 0.25 0.5
0 0.5 0 0.25 0.5
--
+
++
---l-i-+
-
-
-
_
-
6
7
6
9
10
-
-
111213
-
+
mM
+
1415
Fig. 2. Autolytic activation of intracellular p-calpain in rabbit erythrocytes and fusion by treatment with A& and/or CaZ+. The reaction was stopped after 30 min (lanes l-5), 60 min (lanes 6The other procedures were the same 101, or 150 min (lanes 11-15). as in Fig. 1.
Vol.
182,
No.
2, 1992
BIOCHEMICAL
AND
-+-+++ --+++-
ca2+ A2C
-i-i++ --+++----ii
A23167----++
fusion
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-+-+++ --+++----ii
- -
-
-
+
-
-
_
-
-
+
_
1 2
3
4
5
6
7
6
9 10 11 12
-
_
-
+
13 141516
+
-
17 16
Fig. 3. Autolytic activation of intracellular u-calpain in human erythrocytes and fusion by treatment with AzC and/or CaZ+. Erythrocytes were preincubated in the presence (lanes 5, 6, 11, 12, and 18) or absence (lanes l-4, 7-10, and 13-16) of 20 PM 17, A23187 at 37'C for 5 min. The reaction was stopped after 120 min (lanes l-6), 180 min (lanes 7-12), or 240 min (lanes 13-18). The other procedures were the same as in Fig. 1.
still accompanied by fusion (Fig.3). Rabbit erythrocytes gave the same results as human erythrocytes (data not shown). Effect of a protease inhibl'tor, ZLLLal, on AzC-induced fusl*on: To see whether p-calpain activation is necessary for fusion, a protease inhibitor, ZLLLal, was added to the fusion system (Fig.4.). ZLLLal is known to inhibit the autolytic activation of intracelluis increased by the lar p-calpain in erythrocytes when Ca Z+-influx At a concentration of 100 PM, ZLLLal was presence of A23187 (5).
ZLLLal
-+-+++ --++++ ----ioioo
fusion
-
Ca2+ A2C
-
123456
-+-+++ --++++ --
-
-
-
-
-+-+++ --++++ ---
-ioioo
-
-
-
+
7
6
9
10
+_
-
-
11 12
13
-
-ioioopd
-
14 15
+
+_ -
16 1716
Fig. 4. Effect of an exogenously added protease inhibitor, ZLLLal, on fusion and the intracellular autolytic activation of u-calpain in rabbit erythrocytes. Erythrocytes were incubated for 60 min (lanes l-6), 150 min (lanes 7-12) or 180 min (lanes 13-18) in the presence or absence of 0.5 mM CaClz and/or 0.25 pi/ml AZC and/or protease inhibitor at the concentrations indicated. 943
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 5. Micrographs showing A,C-induced fusion of rat erythrocytes. Erythrocytes were incubated as described for Fig. 1. Morphological changes in the erythrocytes were observed under a phase-contrast microscope. (A), Caz+ only; (B), A2C and 1 mM EGTA; (C), AzC and Caz+; (D), A,C, CaZ+ and 10 PM ZLLLal.
found to inhibit the autolytic activation of fusion CaZ+ completely. Simultaneously, (Fig.5-D), showing that fusion is catalyzed p-calpain.
p-calpain was also by the
by AzC and inhibited action of
DISCUSSION Autolytic activation of p-calpain was detected during erythrocyte fusion induced by a fusogen, A2C. In a previous study (5), erythrocyte shape change induced by an increase in Cal+-influx was At the same accompanied by the autolytic activation of u-calpain. time, degradation of membrane proteins was found to occur. Similarly, p-calpain undergoes autolytic activation during erythrocyte fusion, a phenomenon that accompanies shape change. Kosower et al. showed that fusibility is correlated with the net activity of calcium-activated cytoplasmic proteases, with the balance between the protease and its inhibitor, and with the degradation of membrane proteins (6,14); thus, our results support and extend the 944
Vol.
182, No. 2, 1992
BIOCHEMICAL A&Ca2+
AND BIOPHYSICAL
or A23187-Ca2+
RESEARCH COMMUNICATIONS
inhfbitor
p-calpain autolysis
degradation of membrane proteins -
Fig. 6. throcyte
The role of p-calpain membrane.
A2C
in the AzC-induced
fusion
of ery-
previous findings. Although fusion occurs in rat, rabbit, and human erythrocytes after different incubation times, u-calpain is always activated simultaneously with fusion. In erythrocytes preincubated with A23181, both autolytic activation of u-calpain and fusion start early. From these results the scheme outlined in Fig.6 was derived. That is, AzC, like ionophores, promotes the entry of calcium into cells. In response, p-calpain is autolytically activated beneath the plasma membrane and degrades membrane proteins. It is suggested that when AzC approaches protein-free lipid areas of the membrane as micelles, membrane fusion occurs. In addition to the acceleration of fusion by A23187, the inhibition of fusion and the autolytic activation of u-calpain by ZLLLal, a very strong and membrane permeable inhibitor of u-calsupports the idea that calpain is involved in membrane pain, fusion. We believe that this inhibitor provides a powerful tool with which the roles of p-calpain in cells can be analyzed. Finally, membrane fusion plays important roles in a variety of biological processes such as fertilization, muscle development, giant cell formation, exocytosis, endocytosis, and the intracellular exchange of membranes between organelles, for which the Further involvement of Caz+ has been reported (15,16,17,18,19). studies are necessary to expand the results presented here to other fusion-related phenomena to determine whether the autolytic activation of p-calpain is a universal requirement. REFERENCES 1. Suzuki, 2. Melloni,
Trends Biochem. Sci. K. (1987) E., and Pontremoli, S. (1989)
438-444.
945
12, 103-105. Trends Neurosci.
12,
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
3. Ohno, S., Emori, Y., Imajoh, and Suzuki, K. (1984) Nature 4. Murakami, T., Hatanaka, M., /Tokyo/
90,
S.,
RESEARCH COMMUNICATIONS
Kawasaki,
312,
H.,
Kisaragi,
M.,
566-570.
and Murachi,
T.
(1981)
J.
Biochem.
1809-1816.
5. Hayashi, M., Inomata, M., Saito, Y., Ito, H., and Kawashima, S. (1991) Biochim. B~'ophys. Acta 1094, 249-256. 6. Kosower, N.S., Glaser, T., and Kosower, E.M. (1983) Proc. Natl.
Acad.
7. Ito,
A.,
Phaznz.
8. Kosower,
Bull.
12. 13.
USA
E.M.,
23,
80,
R.,
7542-7546.
Miura,
C.,
and Baba,
Y.
(1975)
3106-3113.
Biophys.
Kosower, N.S., and Wegman, P. (1977) 471, 311-329. U.K. (1970) Nature 227, 680-685. M., Kasai, Y., Nakamura, M., and Kawashima,
J.
Chem. 263,
Cbem.
Biochim.
Acta
9. Laemmli, 10. Inomata, 11.
Sci.
Takahashi,
Biol.
S. (1988)
19783-19787.
Kasai, Y., Inomata, M., Hayashi, M., Imahori, K., and Kawashima, S. (1986) J. B1*ochem. (Tokyo) 100, 183-190. Tavassoli, M., Kosower, N.S., Halverson, C., Aoki, M., and E.M. (1980) Biochim. Biophys. Acta 610, 544-558. Kosower, Croall, D.E., and DeMartino, G.N. (1983) J. Bl.01. Chem. 258, 5660-5665.
14.
15.
Glaser, Ridgway, Natl.
16.
17. 18.
T., and Kosower, N.S. (1986) FIBS Lett. 206, 115-120. E.B., Gilkey, J.C., and Jaffe, L.F. (1977) Pro.
Acad.
Sci.
USA
74,
623-627.
couch, C.B., and Strittmatter, W.J. (1983) Cell 32, 257-265. Burgoyne, R.D. (1984) Bl.ocbim. B.z'ophys. Acta 779, 202-216. Ceccarelli, B., and Hurlbut, W.P. (1980) J. Cell Biol. 87, 297-303.
19. Altstiel,
L.,
and Branton,
D. (1983)
946
Cell
32,
921-929.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 939-946
CALPAIN ACTIVATION IS ESSENTIAL FOR MEMBRANE FUSION OF ERYTHROCYTES IN THE PRESENCE OF EXOGENOUS CA2+ Masami
Hayashi,
Yumiko
Saito
and Seiichi
Kawashima
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173, Japan Received December 20, 1991
Summary: The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis8-(2-octylcyclopropyl)octanoate (AK) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Caz+. Under these conditions, the high sensitivity form of calciumprotease (p-calpain) in activated neutral erythrocytes is p-Calpain is activated in accordance activated autolytically. that is, both erythrocyte fusion and autolytic with fusion; activation of u-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A& and Caz+. When erythrocytes are preincubated with the Caz+ ionophore A23187, both fusion and autoA leupeptin analogue, Cbz-Leulytic activation start earlier. Leu-Leu-aldehyde (ZLLLal)! inhibits both the autolytic activation of p-calpain and fusion induced by A;?C and Caz+. These results indicate that treatment of erythrocytes with AzC and Caz+, results in first an influx of Caz+ into the cells, followed by autolytic activation of u-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes. 0 1992 ncademic
Press,
Inc.
Calcium-activated neutral protease (calpain) is an intracellular thiol proteinase that is activated by calcium ions and inhibited by a specific endogenous inhibitor (calpastatin) (1,2). In most cases, there are two forms of calpain, namely, low calcium ion (PM concentrations)-requiring p-calpain and high calcium ion (mM concentrations)-requiring m-calpain. Although the primary structures of the two forms of calpain have been determined from their cDNA sequences (3), their role in cells remains unclear. . . Abbrevlatlons: SDS, sodium dodecyl sulfate; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of SDS; ZLLLal, benzyloxycarbonyl-leucyl-leucyl-leucine aldehyde; A2C, 2-(methoxyethoxy) ethyl-cis-8-(2-octylcyclopropyl)octanoate; kDa, kilo-daltons.
939
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the activation process appears to From many experiments in vitro, include autolysis in the presence of Caz+ during which the N-terminal portions of the large and small subunits are removed (1). Because the intracellular Caz+ concentration is very low, it is process is also not certain whether this activation in vl.tro Accordingly, we chose erythrocytes as model realized in viva. cells since they are the simplest cells and contain only ucalpain (4). In a previous study (5), we reported that erythrocytes undergo a change in shape from a biconcave discocytes to echinocytes and spherocytes upon treatment with ionophore A23187 and Caz+. At the same time, intracellular p-calpain is autolyzed. both the autolytic activation of u-calpain and the Furthermore, shape change induced by A23187-CaZ+ treatment are inhibited by a leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde cell-permeable (ZLLLal), that inhibits the caseinolytic activity of calpain in vl*tro very strongly. Recently, Kosower et al. reported that the fusion of erythrocytes promoted by reagents such as A2C results from an attack by calpain on membrane proteins (6). The fusion of erythrocytes could provide a very useful system in which to study the function of p-calpain in cells. In the present paper, we describe the relationship between erythrocyte fusion and the autolytic activation of p-calpain and the simultaneous inhibition of fusion and calpain activation by ZLLLal. MATERIALS AND METHODS Ionophore A23187 and AzC were purchased from Sigma Co. The protease inhibitor ZLLLal was synthesized to the method of Ito et al. (7). Erythrocytes: Heparinized blood from rats, rabbits, and healthy humans was centrifuged. Plasma and buffy coat were removed and the erythrocytes were washed three times with phosphate-buffered saline (Dulbecco's). Fusion: Erythrocytes were resuspended in buffer (pH 5.6) containing sodium acetate (75 mM), NaCl (70 mM), CaClz (1 mM) and Dextran T-70 (100 mg/ml; Pharmacia) to a hematocrit of 8 - 10% and mixed with an equal volume of AzC suspension (0.5 pi/ml) in NaCl (150 mM) prepared by sonication for 30 set (8). The cell suspensions were incubated at 37'C. Aliquots were removed at intervals for direct observation by phase-contrast microscopy or boiled in an equal volume of sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (9). Analyses of proteins and autolysis of p-calpain: After SDSPAGE, proteins were transferred from the gels to a nitrocellulose membrane. The blotted proteins were incubated with monoclonal anti-calpain antibodies and calpain was detected using horseradish peroxidase-conjugated anti-mouse IgG and diaminobenzidine. A monoclonal antibody, lAsAz, against the large subunit of rabbit u-calpain was used for rabbit and human erythrocytes, while which recognizes the large subunits of both p-calpain and lD=oA,, m-calpain from all animal species, was used for rat erythrocytes Materials:
Chemical according
940
Vol.
182,
No.
2, 1992
because only high affinity
BIOCHEMICAL
AND
1D10A7 among our with rat u-calpain
BIOPHYSICAL
monoclonal (10,ll).
RESEARCH
antibodies
COMMUNICATIONS
reacts
with
RESULT Fmdbl*lity
of
erythrocytes
and autolytic activation al. (12), fusion in rat
ofp-calpain:
As reported by Kosower et erythrocytes is induced by A2C in the presence of Caz+ at 37'C within 30 min. We have previously found that p-calpain is activated autolytically when rabbit erythrocytes are treated with the ionophore A23187 and Ca2+ (5). As shown in Fig.l., the autolytic activation of u-calpain is induced at 30 min only when rat erythrocytes are treated with both A2C and Caz+. The molecular weight of the large subunit of rat p-calpain is 77 kDa, slightly smaller than rabbit or human which are 79 kDa (13). u-calpains, The autolysis of the rat u-calpain large subunit produces a 75 kDa species directly, whereas, the autolytic conversion of the large subunit of rabbit p-calpain is from 79 kDa to 76 kDa via a 77 kDa intermediate. We examined the specificity of our monoclonal antibodies and found that the large subunits of rabbit and human p-calpain are very similar in their reactivities. Since the monoclonal antibody
Ca2+
-+-+
A2C
-
113K
w
88~
e
fusion
-+-+ -
+
+
-
-
+
+
-1 -
-
12
-
-
+
3 4
-
-
587
-
77K 75K
+
8
Fiq. 1. Autolytic activation of intracellular p-calpain in rat erythrocytes and fusion by treatment with A,C and/or Ca2+. Erythrocytes were incubated in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of 0.5 mM CaCl= and in the presence (lanes 3, 4, 7, and 8) or absence (lanes 1, 2, 5, and 6) of 0.25 pi/ml AK. The reaction was started by incqbation at 37'C and stopped after 30 min (lanes l-4) or 60 min (lanes 5-8) by the addition of an equal volume of SDS-sample buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blotted proteins were incubated with an anti-calpain monoclonal antibody, 1D,,A7, and visualized using peroxidase-conjugated anti-mouse IgG and diaminobenzidine. 941
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
raised against the rabbit u-calpain nizes rat p-calpain only slightly, reacts with both u- and m-calpains was used for rat u-calpain. When in the presence of A2C and Ca2+ At this point, started to lyse. continued and the large subunit of and was no longer MW-products antibody. Differences
in
rates
fUSl*On
of
RESEARCH COMMUNICATIONS
large subunit (1AeA2) recoga monoclonal antibody that in all animal species (1DIOA7) rat erythrocytes were incubated for 60 min, the erythrocytes the autolysis of p-calpain u-calpain was degraded to lowrecognized by the monoclonal
erythrocytes
frOn2
Varl*OUS
species:
The time required for fusion in the presence of A2C and Ca2+ varies depending on the animal species. While rat erythrocytes start to fuse after 30 min, rabbit erythrocytes require 150 min and human erythrocytes 240 min (Fig.3). for fusion (Fig.Z), Autolytic activation of p-calpain starts at the same time as fusion (Figs.2 and 3). Effect of A.23187 on A.&-induced fusl*on: In previous studies we have found that p-calpain is activated and degrades cytoskeletal proteins when Ca I+-influx is increased by the addition of the Caz+ ionophore A23187 (5). In order to establish whether Caz+-influx affects the fusion process, we added A23187 to the fusion system. Human erythrocytes were preincubated with A23187 and fusion was started by the addition of A2C and CaZ+. In this case, autolytic activation began earlier than in the absence of A23187, but was
Ca*+
0 0.5
A2C
--+
fusion
_
0 0.25 0.5 ++
-
12345
-
-
-
0 0.5
0 0.25 0.5
0 0.5 0 0.25 0.5
--
+
++
---l-i-+
-
-
-
_
-
6
7
6
9
10
-
-
111213
-
+
mM
+
1415
Fig. 2. Autolytic activation of intracellular p-calpain in rabbit erythrocytes and fusion by treatment with A& and/or CaZ+. The reaction was stopped after 30 min (lanes l-5), 60 min (lanes 6The other procedures were the same 101, or 150 min (lanes 11-15). as in Fig. 1.
Vol.
182,
No.
2, 1992
BIOCHEMICAL
AND
-+-+++ --+++-
ca2+ A2C
-i-i++ --+++----ii
A23167----++
fusion
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-+-+++ --+++----ii
- -
-
-
+
-
-
_
-
-
+
_
1 2
3
4
5
6
7
6
9 10 11 12
-
_
-
+
13 141516
+
-
17 16
Fig. 3. Autolytic activation of intracellular u-calpain in human erythrocytes and fusion by treatment with AzC and/or CaZ+. Erythrocytes were preincubated in the presence (lanes 5, 6, 11, 12, and 18) or absence (lanes l-4, 7-10, and 13-16) of 20 PM 17, A23187 at 37'C for 5 min. The reaction was stopped after 120 min (lanes l-6), 180 min (lanes 7-12), or 240 min (lanes 13-18). The other procedures were the same as in Fig. 1.
still accompanied by fusion (Fig.3). Rabbit erythrocytes gave the same results as human erythrocytes (data not shown). Effect of a protease inhibl'tor, ZLLLal, on AzC-induced fusl*on: To see whether p-calpain activation is necessary for fusion, a protease inhibitor, ZLLLal, was added to the fusion system (Fig.4.). ZLLLal is known to inhibit the autolytic activation of intracelluis increased by the lar p-calpain in erythrocytes when Ca Z+-influx At a concentration of 100 PM, ZLLLal was presence of A23187 (5).
ZLLLal
-+-+++ --++++ ----ioioo
fusion
-
Ca2+ A2C
-
123456
-+-+++ --++++ --
-
-
-
-
-+-+++ --++++ ---
-ioioo
-
-
-
+
7
6
9
10
+_
-
-
11 12
13
-
-ioioopd
-
14 15
+
+_ -
16 1716
Fig. 4. Effect of an exogenously added protease inhibitor, ZLLLal, on fusion and the intracellular autolytic activation of u-calpain in rabbit erythrocytes. Erythrocytes were incubated for 60 min (lanes l-6), 150 min (lanes 7-12) or 180 min (lanes 13-18) in the presence or absence of 0.5 mM CaClz and/or 0.25 pi/ml AZC and/or protease inhibitor at the concentrations indicated. 943
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Fig. 5. Micrographs showing A,C-induced fusion of rat erythrocytes. Erythrocytes were incubated as described for Fig. 1. Morphological changes in the erythrocytes were observed under a phase-contrast microscope. (A), Caz+ only; (B), A2C and 1 mM EGTA; (C), AzC and Caz+; (D), A,C, CaZ+ and 10 PM ZLLLal.
found to inhibit the autolytic activation of fusion CaZ+ completely. Simultaneously, (Fig.5-D), showing that fusion is catalyzed p-calpain.
p-calpain was also by the
by AzC and inhibited action of
DISCUSSION Autolytic activation of p-calpain was detected during erythrocyte fusion induced by a fusogen, A2C. In a previous study (5), erythrocyte shape change induced by an increase in Cal+-influx was At the same accompanied by the autolytic activation of u-calpain. time, degradation of membrane proteins was found to occur. Similarly, p-calpain undergoes autolytic activation during erythrocyte fusion, a phenomenon that accompanies shape change. Kosower et al. showed that fusibility is correlated with the net activity of calcium-activated cytoplasmic proteases, with the balance between the protease and its inhibitor, and with the degradation of membrane proteins (6,14); thus, our results support and extend the 944
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or A23187-Ca2+
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inhfbitor
p-calpain autolysis
degradation of membrane proteins -
Fig. 6. throcyte
The role of p-calpain membrane.
A2C
in the AzC-induced
fusion
of ery-
previous findings. Although fusion occurs in rat, rabbit, and human erythrocytes after different incubation times, u-calpain is always activated simultaneously with fusion. In erythrocytes preincubated with A23181, both autolytic activation of u-calpain and fusion start early. From these results the scheme outlined in Fig.6 was derived. That is, AzC, like ionophores, promotes the entry of calcium into cells. In response, p-calpain is autolytically activated beneath the plasma membrane and degrades membrane proteins. It is suggested that when AzC approaches protein-free lipid areas of the membrane as micelles, membrane fusion occurs. In addition to the acceleration of fusion by A23187, the inhibition of fusion and the autolytic activation of u-calpain by ZLLLal, a very strong and membrane permeable inhibitor of u-calsupports the idea that calpain is involved in membrane pain, fusion. We believe that this inhibitor provides a powerful tool with which the roles of p-calpain in cells can be analyzed. Finally, membrane fusion plays important roles in a variety of biological processes such as fertilization, muscle development, giant cell formation, exocytosis, endocytosis, and the intracellular exchange of membranes between organelles, for which the Further involvement of Caz+ has been reported (15,16,17,18,19). studies are necessary to expand the results presented here to other fusion-related phenomena to determine whether the autolytic activation of p-calpain is a universal requirement. REFERENCES 1. Suzuki, 2. Melloni,
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