- Email: [email protected]
Proteolysis of highly polysialylated NCAM by the tissue plasminogen activator-plasmin system in rats
Neuroscience Letters 246 (1998) 37–40
Proteolysis of highly polysialylated NCAM by the tissue plasminogen activator-plasmin system in rats Akira Endo a, Nobuo Nagai b, Tetsumei Urano b, Hayato Ihara b, Yumiko Takada c, Kenji Hashimoto a, Akikazu Takada b ,* a
Department of Dentistry and Oral and Maxilloralfacial Surgery, Hamamatsu University School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan b Department of Physiology, Hamamatsu University, School of Medicine, 3600 Handa-cho, Hamamatsu-shi, Shizuoka-ken, 431-3192 Japan c Department of Pathophysiology, Hamamatsu University School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan Received 25 December 1997; received in revised form 5 March 1998; accepted 6 March 1998
Abstract Tissue-type plasminogen activator (tPA), a serine protease which converts the zymogen plasminogen to the active protease plasmin, is believed to regulate neurite extension and neural cell migration by modulating extracellular metabolism. The highly polysialylated form of the neural cell adhesion molecule (NCAM-H) is strongly expressed in the developing brain and is believed to play a role in organizing the neural network. In this report, we incubated neonatal rat brain homogenates with human tPA and rat plasminogen in order to determine whether NCAM-H would be degraded. NCAM-H was degraded by plasmin which was formed from rat plasminogen by human tPA. The degradation was inhibited by the addition of plasminogen activator inhibitor type 1 (PAI-1) or aprotinin. These results suggest a possible contribution of the tPA-plasmin system to NCAM-H turnover in the developing brain. 1998 Elsevier Science Ireland Ltd.
Keywords: Development; Extracellular matrix; Tissue-type plasminogen activator; Plasmin; Plasminogen; Neural cell adhesion molecule; Proteolysis
Tissue-type plasminogen activator (tPA) is a serine protease which converts the zymogen plasminogen to the active broad-spectrum protease plasmin. Plasmin degrades fibrin and other extracellular proteins. In addition, plasmin is one of the activators of metalloprotease precursors and could play a pivotal role in extracellular matrix (ECM) degradation. Neural development requires the migration of neurons and neurite outgrowth, and is a complex phenomenon that requires multiple interactions between the growth cone, the growing neuronal membrane and certain components of the ECM. The extension and retraction of microspikes, and their transitory contacts with their surroundings require alternating phrases of adhesion and de-adhesion. In the central nervous system, it has been reported that tPA mRNA is * Corresponding author. Tel.: +81 53 4352247; fax: +81 53 4349225; e-mail: [email protected]
highly expressed in the developing brain [4] and increased tPA activity was detected at the level of the growth cone. tPA is believed to regulate the transient interactions of the growth cone with its environment. Therefore, tPA is thought to be involved in the migration of granule neurons [7], elimination of synapses and neurite outgrowth [9] by modulating cell/cell and cell/matrix interactions [10]. On the other hand, the neural cell adhesion molecule (NCAM) is extensively expressed in the central nervous system. The highly polysialylated NCAM (NCAM-H) is an embryonic form of NCAM which is transiently expressed in the central nervous system from the late embryonic stage to the early postnatal stage and is believed to be involved in the organization of the neural network [12]. Therefore, it is possible that NCAM-H is a substrate for extracellular proteolysis by the tPA-plasmin system in the developing brain. In this study, we investigated this possibility by determining whether NCAM-H is degraded by the tPA-plasmin system.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00204- 3
38
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
Postnatal 6 day (P6) and 10-week-old Wistar rats were used in these experiments. The P6 rats were perfused with 10 ml of saline under anesthesia with ether and their brains were quickly removed. The 10-week-old rats were also perfused with saline under anesthesia with pentobarbital, and their brains and kidneys were quickly removed. The brains and kidneys were homogenized in 10 volumes of Tris buffer (pH 7.4) containing 0.1% Triton X-100 and kept at −80°C until used for the experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [6]. These homogenates were mixed with one third volume of SDS sample buffer (8% SDS, 40% glycerin and 0.1% bromophenol blue in 0.25 M Tris–HCl (pH 6.8) and were applied to the SDS gel at 30 mg of protein in each lane. Fibrin autography was performed as previously described [1]. Briefly, after washing with 2.5% Triton X-100 for 15 min, SDS gels were laid carefully over fibrin indicator gels which had the following composition (final concentration): 60 mM Tris (pH 7.4), 17.5 mM NaCl, 8 mg/ml of agarose, 2 mg/ml of human fibrinogen (Enzyme Research Laboratories), 0.06 U/ml of human thrombin, and 4 mg/ml of human Glu-plasminogen. Human Glu-plasminogen was purified as previously described [3]. Incubations were performed in a moist chamber at 37°C for 24 h. In order to determine whether NCAM-H could be a substrate for extracellular proteolysis by the tPA-plasmin system, neonatal rat brain homogenates with 10 mM EDTA, which was added to chelate Ca2 + , were incubated at 37°C in the presence or absence of 2 mM rat plasminogen, 70 nM human purified tPA (kindly provided by Daiichi Seiyaku, Tokyo, Japan and Sumitomo Seiyaku, Osaka, Japan) and 70 nM plasminogen activator inhibitor type 1 (PAI-1). Rat plasminogen was purified as previously described [3]. Active human PAI-1 was purified as previously described [5]. We observed the degradation of NCAM-H in the presence of both plasminogen and tPA. Then, in order to investigate the sequential degradation of NCAM-H by the tPA-
Fig. 1. Fibrin autography of the neonatal brain, adult brain and adult kidney homogenates. Lane 1, purified rat tPA; lane 2, adult rat brain homogenate; lane 3, P6 rat brain homogenate; lane 4, adult rat kidney homogenate. The kidney was used as a standard which contains both tPA and uPA [5]. The position of tPA and uPA is shown on the right side.
Fig. 2. Degradation of NCAM-H by the tPA-plasmin system in vitro. Immunoblot analysis of NCAM-H was performed in the neonatal rat brain homogenates. The homogenates were incubated with 10 mM EDTA in the presence or absence of 70 nM human tPA, 2 mM rat plasminogen and 70 nM PAI-1 for 240 min at 37°C. Immunoblot analysis was developed with the anti-rat NCAM-H IgM which was a gift from Dr. Tatsunori Seki (Juntendo University School of Medicine).
plasmin system, we incubated samples at 37°C for 10 min, 30 min, 60 min and 240 min. In order to determine the direct contribution of the tPA-plasmin system to NCAM-H degradation, incubations were performed for 240 min with 70 nM PAI-1 which is an inhibitor of plasminogen activators (PAs) and 200 unit/ml of aprotinin (final concentration) which is a protease inhibitor with a specificity for plasmin. Following each incubation, samples of 8 mg were treated with one third volume of SDS sample buffer (8% SDS, 40% glycerin, 20% mercaptoethanol, and 0.1% bromophenol blue in 0.25 M Tris–HCl (pH 6.8)), and kept overnight at 4°C and then separated by SDS-PAGE. Separated proteins were then transferred to nitrocellulose membranes according to Towbin et al. [14]. After blocking with 10% commercial non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS) for 1 h, these membranes were then treated overnight with an anti-rat NCAM-H IgM at a 1:2000 dilution with TTBS, followed by the ABC method with a Vectastain kit [12]. Development of the immunoblot was performed with an ECL kit (Amersham, UK) on X-ray film. Zymography was used to assess the levels of tPA activity in adult and neonatal brain homogenates (Fig. 1). In the lane of the kidney homogenate, the activities of tPA as well as urokinase-type plasminogen activator (uPA) were observed [11]. While in neonatal (P6) and adult brain homogenates, only tPA activity was observed. The tPA activity was more strongly expressed in the neonatal brain homogenate than in the adult brain homogenate. The results showed that the activity of tPA in the brain is higher during the neonatal than the adult stage. As shown in Fig. 2, NCAM-H in neonatal rat brain homogenates containing 10 mM EDTA was degraded when incubated with human tPA and rat plasminogen. After a 240-min
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
39
Fig. 3. Time course of NCAM-H degradation by the tPA-plasmin system. (A) Immunoblot analysis of NCAM-H was performed in the neonatal rat brain homogenates. The homogenates were incubated with 10 mM EDTA in the presence of both 70 nM human tPA and 2 mM rat plasminogen for 10 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 240 min (lane 5). The control sample (lane 1) was incubated for 0 min without the addition of tPA and plasminogen. (B) Samples were also incubated for 240 min in the presence of both 70 nM human tPA and 2 mM rat plasminogen (lane 1) or with the addition of either 70 nM PAI-1 (lane 2) or 200 unit/ml of aprotinin (final concentration; lane 3). The immunoblots were developed with the anti-rat NCAM-H IgM.
incubation, in the presence of both tPA and plasminogen, NCAM-H was degraded. In the absence of plasminogen, NCAM-H was degraded very slightly even if tPA was added. When we incubated the sample with plasminogen alone, NCAM-H was also degraded because of the endogenous tPA but the amount of degradation was small compared to the sample incubated with both tPA and plasminogen. Because we used brain homogenate at a 50-fold dilution in these experiments, the concentration of tPA in the sample was very low. When we incubated the sample with plasminogen and PAI-1, the degradation of NCAM-H was inhibited. These results show that NCAM-H was degraded in vitro by the activation of the tPA-plasmin system. Fig. 3A shows the time dependency of the degradation of NCAM-H in the brain homogenate during incubation in the presence of tPA and plasminogen with 10 mM EDTA. The degradation of NCAM-H occurred within 1 h. Considering that the antibody used in this experiment recognizes the polysialated site of NCAM-H and the bands of approximately 90 and 60 kDa shown in Fig. 3 were not observed in the absence of plasminogen (lane 1), these bands might be due to a reaction with the glycosylation site of plasminogen and plasmin. In order to examine this possibility, we incubated tPA and plasminogen without the brain homogenate and observed bands of approximately 90 and 60 kDa (data not shown). These bands showed that the conversion of plasminogen to plasmin is time dependent, i.e. about half of the added plasminogen was converted to plasmin after 1 h and most of NCAM-H was degraded. The evidence showed that the degradation of NCAM-H was in parallel with the formation of plasmin. PAI-1 is a inhibitor of PAs and aprotinin is a protease inhibitor with a high specificity for plasmin. As shown in Fig. 3B, after the addition of either of them, the degradation of NCAM-H was inhibited. These
results suggest that activation of plasmin by tPA is essential for the degradation of NCAM-H. In the central nervous system, plasminogen appears to be secreted from glia [8] but its extracellular concentration is unknown. In this experiment, we used brain homogenates at a 50-fold dilution. Furthermore, the extracellular space in the brain is believed to be about 10–15% of the total brain volume [2]. Therefore, the physiological extracellular concentration of plasminogen in the brain is estimated to be about 333–500 times higher than that used in this experiment, which might be enough to degrade NCAM-H when it is processed by the secreted tPA. NCAM-H was expressed in the central nervous system during the late embryonic and early postnatal stages [12] when tPA activity in the central nervous system was high [13]. From our results that NCAM-H is degraded by the tPA-plasmin system, it is suggested that the tPA-plasmin system acts as a mechanism for the degradation of NCAMH in the developing brain. Considering that NCAM-H participated in axonal growth and pathfinding [15] and tPA is thought to be involved in the migration of neurons, elimination of synapses and neurite outgrowth by modulating cell/ cell and cell/matrix interactions, the tPA-plasmin system may contribute to the organization of neural network through the regulation of NCAM-H turnover in the developing brain. We thank Dr. Tatsunori Seki of the Juntendo University School of Medicine for the gift of the anti-rat NCAM-H antibody. [1] Brunner, G. and Schirrmacher, V., Fibrin autography of plasminogen activator by electrophoretic transfer into fibrin agar gels, Anal. Biochem., 168 (1988) 411–416. [2] Davson, H. and Bradbury, M., The extracellular space of the brain, Prog. Brain Res., 15 (1965) 124–134.
40
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
[3] Deutsch, D.G. and Mertz, E.T., Plasminogen: purification from human plasma by affinity chromatography, Science, 170 (1970) 1095–1096. [4] Friedman, G.C. and Seeds, N.W., Tissue plasminogen activator expression in the embryonic nervous system, Dev. Brain Res., 81 (1994) 41–49. [5] Lawrence, D., Strandberg, L., Grundstro¨m, T. and Ny, T., Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells, Eur. J. Biochem., 186 (1989) 523–533. [6] Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680–685. [7] Moonen, G., Grau, W.M. and Selak, I., Plasminogen activatorplasmin system and neuronal migration, Nature, 298 (1982) 753–755. [8] Nakajima, K., Tsuzaki, N., Nagata, K., Takemoto, N. and Kohsaka, S., Production and secretion of plasminogen in cultured rat brain microglia, FEBS Lett., 308 (1992) 179–182. [9] Pittman, R.N., Ivins, J.K. and Buettner, H.M., Neuronal plasmi-
[10]
[11]
[12]
[13]
[14]
[15]
nogen activators: cell surface binding sites and involvement in neurite outgrowth, J. Neurosci., 9 (1989) 4269–4286. Sappino, A.P., Madani, R., Huarte, J., Belin, D., Kiss, J.Z., Wohlwend, A. and Vassalli, J.D., Extracellular proteolysis in the adult murine brain, J. Clin. Invest., 92 (1993) 679–685. Sappino, A.P., Huarte, J., Vassalli, J.D. and Belin, D., Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney, J. Clin. Invest., 87 (1991) 962–970. Seki, T. and Arai, Y., Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat, Anat. Embryol., 184 (1991) 395–401. Soreq, H. and Miskin, R., Plasminogen activator in the developing rat cerebellum: biosynthesis and localization in granular neurons, Brain Res., 313 (1983) 149–158. Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 (1979) 4350–4354. van den Pol, A.N. and Kim, W.T., NILE/L1 and NCAM-polysialic acid expression on growing axons of isolated neurons, J. Comp. Neurol., 332 (1993) 237–257.
Proteolysis of highly polysialylated NCAM by the tissue plasminogen activator-plasmin system in rats Akira Endo a, Nobuo Nagai b, Tetsumei Urano b, Hayato Ihara b, Yumiko Takada c, Kenji Hashimoto a, Akikazu Takada b ,* a
Department of Dentistry and Oral and Maxilloralfacial Surgery, Hamamatsu University School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan b Department of Physiology, Hamamatsu University, School of Medicine, 3600 Handa-cho, Hamamatsu-shi, Shizuoka-ken, 431-3192 Japan c Department of Pathophysiology, Hamamatsu University School of Medicine, Hamamatsu-shi, Shizuoka-ken, Japan Received 25 December 1997; received in revised form 5 March 1998; accepted 6 March 1998
Abstract Tissue-type plasminogen activator (tPA), a serine protease which converts the zymogen plasminogen to the active protease plasmin, is believed to regulate neurite extension and neural cell migration by modulating extracellular metabolism. The highly polysialylated form of the neural cell adhesion molecule (NCAM-H) is strongly expressed in the developing brain and is believed to play a role in organizing the neural network. In this report, we incubated neonatal rat brain homogenates with human tPA and rat plasminogen in order to determine whether NCAM-H would be degraded. NCAM-H was degraded by plasmin which was formed from rat plasminogen by human tPA. The degradation was inhibited by the addition of plasminogen activator inhibitor type 1 (PAI-1) or aprotinin. These results suggest a possible contribution of the tPA-plasmin system to NCAM-H turnover in the developing brain. 1998 Elsevier Science Ireland Ltd.
Keywords: Development; Extracellular matrix; Tissue-type plasminogen activator; Plasmin; Plasminogen; Neural cell adhesion molecule; Proteolysis
Tissue-type plasminogen activator (tPA) is a serine protease which converts the zymogen plasminogen to the active broad-spectrum protease plasmin. Plasmin degrades fibrin and other extracellular proteins. In addition, plasmin is one of the activators of metalloprotease precursors and could play a pivotal role in extracellular matrix (ECM) degradation. Neural development requires the migration of neurons and neurite outgrowth, and is a complex phenomenon that requires multiple interactions between the growth cone, the growing neuronal membrane and certain components of the ECM. The extension and retraction of microspikes, and their transitory contacts with their surroundings require alternating phrases of adhesion and de-adhesion. In the central nervous system, it has been reported that tPA mRNA is * Corresponding author. Tel.: +81 53 4352247; fax: +81 53 4349225; e-mail: [email protected]
highly expressed in the developing brain [4] and increased tPA activity was detected at the level of the growth cone. tPA is believed to regulate the transient interactions of the growth cone with its environment. Therefore, tPA is thought to be involved in the migration of granule neurons [7], elimination of synapses and neurite outgrowth [9] by modulating cell/cell and cell/matrix interactions [10]. On the other hand, the neural cell adhesion molecule (NCAM) is extensively expressed in the central nervous system. The highly polysialylated NCAM (NCAM-H) is an embryonic form of NCAM which is transiently expressed in the central nervous system from the late embryonic stage to the early postnatal stage and is believed to be involved in the organization of the neural network [12]. Therefore, it is possible that NCAM-H is a substrate for extracellular proteolysis by the tPA-plasmin system in the developing brain. In this study, we investigated this possibility by determining whether NCAM-H is degraded by the tPA-plasmin system.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00204- 3
38
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
Postnatal 6 day (P6) and 10-week-old Wistar rats were used in these experiments. The P6 rats were perfused with 10 ml of saline under anesthesia with ether and their brains were quickly removed. The 10-week-old rats were also perfused with saline under anesthesia with pentobarbital, and their brains and kidneys were quickly removed. The brains and kidneys were homogenized in 10 volumes of Tris buffer (pH 7.4) containing 0.1% Triton X-100 and kept at −80°C until used for the experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [6]. These homogenates were mixed with one third volume of SDS sample buffer (8% SDS, 40% glycerin and 0.1% bromophenol blue in 0.25 M Tris–HCl (pH 6.8) and were applied to the SDS gel at 30 mg of protein in each lane. Fibrin autography was performed as previously described [1]. Briefly, after washing with 2.5% Triton X-100 for 15 min, SDS gels were laid carefully over fibrin indicator gels which had the following composition (final concentration): 60 mM Tris (pH 7.4), 17.5 mM NaCl, 8 mg/ml of agarose, 2 mg/ml of human fibrinogen (Enzyme Research Laboratories), 0.06 U/ml of human thrombin, and 4 mg/ml of human Glu-plasminogen. Human Glu-plasminogen was purified as previously described [3]. Incubations were performed in a moist chamber at 37°C for 24 h. In order to determine whether NCAM-H could be a substrate for extracellular proteolysis by the tPA-plasmin system, neonatal rat brain homogenates with 10 mM EDTA, which was added to chelate Ca2 + , were incubated at 37°C in the presence or absence of 2 mM rat plasminogen, 70 nM human purified tPA (kindly provided by Daiichi Seiyaku, Tokyo, Japan and Sumitomo Seiyaku, Osaka, Japan) and 70 nM plasminogen activator inhibitor type 1 (PAI-1). Rat plasminogen was purified as previously described [3]. Active human PAI-1 was purified as previously described [5]. We observed the degradation of NCAM-H in the presence of both plasminogen and tPA. Then, in order to investigate the sequential degradation of NCAM-H by the tPA-
Fig. 1. Fibrin autography of the neonatal brain, adult brain and adult kidney homogenates. Lane 1, purified rat tPA; lane 2, adult rat brain homogenate; lane 3, P6 rat brain homogenate; lane 4, adult rat kidney homogenate. The kidney was used as a standard which contains both tPA and uPA [5]. The position of tPA and uPA is shown on the right side.
Fig. 2. Degradation of NCAM-H by the tPA-plasmin system in vitro. Immunoblot analysis of NCAM-H was performed in the neonatal rat brain homogenates. The homogenates were incubated with 10 mM EDTA in the presence or absence of 70 nM human tPA, 2 mM rat plasminogen and 70 nM PAI-1 for 240 min at 37°C. Immunoblot analysis was developed with the anti-rat NCAM-H IgM which was a gift from Dr. Tatsunori Seki (Juntendo University School of Medicine).
plasmin system, we incubated samples at 37°C for 10 min, 30 min, 60 min and 240 min. In order to determine the direct contribution of the tPA-plasmin system to NCAM-H degradation, incubations were performed for 240 min with 70 nM PAI-1 which is an inhibitor of plasminogen activators (PAs) and 200 unit/ml of aprotinin (final concentration) which is a protease inhibitor with a specificity for plasmin. Following each incubation, samples of 8 mg were treated with one third volume of SDS sample buffer (8% SDS, 40% glycerin, 20% mercaptoethanol, and 0.1% bromophenol blue in 0.25 M Tris–HCl (pH 6.8)), and kept overnight at 4°C and then separated by SDS-PAGE. Separated proteins were then transferred to nitrocellulose membranes according to Towbin et al. [14]. After blocking with 10% commercial non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS) for 1 h, these membranes were then treated overnight with an anti-rat NCAM-H IgM at a 1:2000 dilution with TTBS, followed by the ABC method with a Vectastain kit [12]. Development of the immunoblot was performed with an ECL kit (Amersham, UK) on X-ray film. Zymography was used to assess the levels of tPA activity in adult and neonatal brain homogenates (Fig. 1). In the lane of the kidney homogenate, the activities of tPA as well as urokinase-type plasminogen activator (uPA) were observed [11]. While in neonatal (P6) and adult brain homogenates, only tPA activity was observed. The tPA activity was more strongly expressed in the neonatal brain homogenate than in the adult brain homogenate. The results showed that the activity of tPA in the brain is higher during the neonatal than the adult stage. As shown in Fig. 2, NCAM-H in neonatal rat brain homogenates containing 10 mM EDTA was degraded when incubated with human tPA and rat plasminogen. After a 240-min
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
39
Fig. 3. Time course of NCAM-H degradation by the tPA-plasmin system. (A) Immunoblot analysis of NCAM-H was performed in the neonatal rat brain homogenates. The homogenates were incubated with 10 mM EDTA in the presence of both 70 nM human tPA and 2 mM rat plasminogen for 10 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 240 min (lane 5). The control sample (lane 1) was incubated for 0 min without the addition of tPA and plasminogen. (B) Samples were also incubated for 240 min in the presence of both 70 nM human tPA and 2 mM rat plasminogen (lane 1) or with the addition of either 70 nM PAI-1 (lane 2) or 200 unit/ml of aprotinin (final concentration; lane 3). The immunoblots were developed with the anti-rat NCAM-H IgM.
incubation, in the presence of both tPA and plasminogen, NCAM-H was degraded. In the absence of plasminogen, NCAM-H was degraded very slightly even if tPA was added. When we incubated the sample with plasminogen alone, NCAM-H was also degraded because of the endogenous tPA but the amount of degradation was small compared to the sample incubated with both tPA and plasminogen. Because we used brain homogenate at a 50-fold dilution in these experiments, the concentration of tPA in the sample was very low. When we incubated the sample with plasminogen and PAI-1, the degradation of NCAM-H was inhibited. These results show that NCAM-H was degraded in vitro by the activation of the tPA-plasmin system. Fig. 3A shows the time dependency of the degradation of NCAM-H in the brain homogenate during incubation in the presence of tPA and plasminogen with 10 mM EDTA. The degradation of NCAM-H occurred within 1 h. Considering that the antibody used in this experiment recognizes the polysialated site of NCAM-H and the bands of approximately 90 and 60 kDa shown in Fig. 3 were not observed in the absence of plasminogen (lane 1), these bands might be due to a reaction with the glycosylation site of plasminogen and plasmin. In order to examine this possibility, we incubated tPA and plasminogen without the brain homogenate and observed bands of approximately 90 and 60 kDa (data not shown). These bands showed that the conversion of plasminogen to plasmin is time dependent, i.e. about half of the added plasminogen was converted to plasmin after 1 h and most of NCAM-H was degraded. The evidence showed that the degradation of NCAM-H was in parallel with the formation of plasmin. PAI-1 is a inhibitor of PAs and aprotinin is a protease inhibitor with a high specificity for plasmin. As shown in Fig. 3B, after the addition of either of them, the degradation of NCAM-H was inhibited. These
results suggest that activation of plasmin by tPA is essential for the degradation of NCAM-H. In the central nervous system, plasminogen appears to be secreted from glia [8] but its extracellular concentration is unknown. In this experiment, we used brain homogenates at a 50-fold dilution. Furthermore, the extracellular space in the brain is believed to be about 10–15% of the total brain volume [2]. Therefore, the physiological extracellular concentration of plasminogen in the brain is estimated to be about 333–500 times higher than that used in this experiment, which might be enough to degrade NCAM-H when it is processed by the secreted tPA. NCAM-H was expressed in the central nervous system during the late embryonic and early postnatal stages [12] when tPA activity in the central nervous system was high [13]. From our results that NCAM-H is degraded by the tPA-plasmin system, it is suggested that the tPA-plasmin system acts as a mechanism for the degradation of NCAMH in the developing brain. Considering that NCAM-H participated in axonal growth and pathfinding [15] and tPA is thought to be involved in the migration of neurons, elimination of synapses and neurite outgrowth by modulating cell/ cell and cell/matrix interactions, the tPA-plasmin system may contribute to the organization of neural network through the regulation of NCAM-H turnover in the developing brain. We thank Dr. Tatsunori Seki of the Juntendo University School of Medicine for the gift of the anti-rat NCAM-H antibody. [1] Brunner, G. and Schirrmacher, V., Fibrin autography of plasminogen activator by electrophoretic transfer into fibrin agar gels, Anal. Biochem., 168 (1988) 411–416. [2] Davson, H. and Bradbury, M., The extracellular space of the brain, Prog. Brain Res., 15 (1965) 124–134.
40
A. Endo et al. / Neuroscience Letters 246 (1998) 37–40
[3] Deutsch, D.G. and Mertz, E.T., Plasminogen: purification from human plasma by affinity chromatography, Science, 170 (1970) 1095–1096. [4] Friedman, G.C. and Seeds, N.W., Tissue plasminogen activator expression in the embryonic nervous system, Dev. Brain Res., 81 (1994) 41–49. [5] Lawrence, D., Strandberg, L., Grundstro¨m, T. and Ny, T., Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells, Eur. J. Biochem., 186 (1989) 523–533. [6] Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680–685. [7] Moonen, G., Grau, W.M. and Selak, I., Plasminogen activatorplasmin system and neuronal migration, Nature, 298 (1982) 753–755. [8] Nakajima, K., Tsuzaki, N., Nagata, K., Takemoto, N. and Kohsaka, S., Production and secretion of plasminogen in cultured rat brain microglia, FEBS Lett., 308 (1992) 179–182. [9] Pittman, R.N., Ivins, J.K. and Buettner, H.M., Neuronal plasmi-
[10]
[11]
[12]
[13]
[14]
[15]
nogen activators: cell surface binding sites and involvement in neurite outgrowth, J. Neurosci., 9 (1989) 4269–4286. Sappino, A.P., Madani, R., Huarte, J., Belin, D., Kiss, J.Z., Wohlwend, A. and Vassalli, J.D., Extracellular proteolysis in the adult murine brain, J. Clin. Invest., 92 (1993) 679–685. Sappino, A.P., Huarte, J., Vassalli, J.D. and Belin, D., Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney, J. Clin. Invest., 87 (1991) 962–970. Seki, T. and Arai, Y., Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat, Anat. Embryol., 184 (1991) 395–401. Soreq, H. and Miskin, R., Plasminogen activator in the developing rat cerebellum: biosynthesis and localization in granular neurons, Brain Res., 313 (1983) 149–158. Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 (1979) 4350–4354. van den Pol, A.N. and Kim, W.T., NILE/L1 and NCAM-polysialic acid expression on growing axons of isolated neurons, J. Comp. Neurol., 332 (1993) 237–257.