Brain Research, 529 (1990) 91-95
91
Elsevier BRES 15893
Dermatan sulfate and de-sulfated heparin solubilized collagen-tailed acetylcholinesterase from the rat neuromuscular junction Rommy von Bernhardi* and Nibaldo C. Inestrosa Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences and Medicine, Catholic University of Chile, Santiago (Chile) (Accepted 27 March 1990)
Key words: Aeetyleholinesterase; Asymmetric form; Proteoglycan; Skeletal muscle; Synapsis
We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (ACHE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed ACHE. Taking into account that the sohibilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina.
INTRODUCTION
A c e t y l c h o l i n e s t e r a s e (ACHE, E C 3.1.1.7) is a polym o r p h i c enzyme which m a y be classified in globular and asymmetric forms 2. T h e asymmetric forms are concent r a t e d at the n e u r o m u s c u l a r junction and consist of 3 t e t r a m e r s linked to a triple helix tail. T h e enzyme is specifically associated to the basal lamina by the collagenlike tail t h r o u g h proteoglycans 4. Glycosaminoglycans ( G A G s ) are the main c o m p o nents of b a s e m e n t m e m b r a n e proteoglycans. F o r their polyanionic character, G A G s can bind o t h e r molecules, especially cationic amines, p e p t i d e s and proteins, some of which are k n o w n to be essential for cell function 9'12. In particular at cholinergic synapses, h e p a r a n sulfate residues a p p e a r to be associated with acetylcholine r e c e p t o r clusters and with the collagen-tailed A C h E 11. L a t e l y it was shown that heparin, besides releasing asymmetric A C h E from the basal lamina of skeletal muscle, specifically solubilizes a d e r m a t a n sulfate proteoglycan ( D S P G ) 6. This result was the first evidence of the existence of D S P G at the skeletal muscle cells surface and suggested a possible association b e t w e e n asymmetric A C h E and D S P G at the muscle basal lamina.
We r e p o r t here that d e r m a t a n sulfate solubilizes asymmetric A C h E from the muscle basal lamina but only detaches a minimal a m o u n t of the A C h E b o u n d to an h e p a r i n - a g a r o s e column. A d d i t i o n a l e x p e r i m e n t s show that de-sulfated heparin as well as low-molecular weight h e p a r i n also release a s y m m e t r i c A C h E from the rat n e u r o m u s c u l a r junction. MATERIALS AND METHODS The following chemicals were used: heparin from bovine lung and porcine intestinal mucosa (including low molecular weight heparin), heparan sulfate, heparin de-sulfated and chondroitin sulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Heparinagarose was purchased from Bethesda Research Laboratories (Gaithersbnrg, MA). Other reagents, where not specified, were obtained from commercial sources.
Surgical procedures Male Sprague-Dawley rats of 250-300 g were maintained with free access to food and water. Their diaphragms were removed from the ribs, washed and kept in ice-cold saline. The muscles were then divided into endplate and non-endplate regionst3. Only the endplate region were used in this work.
AChE extraction procedure The experiments were performed using sequential extraction procedures. The tissue was weighed, finely minced with scissors and homogenized (1:10 w/v) in low ionic strength buffer, 25 mM
* Present address: Department of Biology, University of Utah, Salt Lake City, UT, U.S.A.
Correspondence: N.C. Inestrosa, Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences and Medicine, Catholic University of Chile, P.O. Box l14-D, Santiago, Chile. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
92 Tris-HCI (pH 7.4), 1% Triton X-100 in the presence of protease inhibitors: 2 mM EDTA, 0.1 mM N-ethylmaleimide. 20 Hg/ml benzamidine, 0.25 mg/ml soy-bean trypsin inhibitor and 1 mg/ml bacitracine. Homogenization was performed in a glass-to-glass homogenizer. The homogenate was centrifuged at 10,000 g during 15 min in an Eppendorf centrifuge, and the solubilized AChE activity was determined in the supernatant. All procedures were conducted at 4 °C. Usually 3-4 sequential extractions were carried out, in order to remove most of the globular AChE forms attached to cellular membranes. After centrifugation, the residual pellet was rehomogenized in the same above buffer twice. The last pellet was resuspended (1:5 w/v) in a low ionic strength buffer and then it was separated in various aliquots that were rehomogenized in Tris buffer containing the different GAGs at required concentration. Finally, the GAG-containingsamples were centrifuged at 10,000 g during 15 rain, and the AChE activity was determined in the supernatant.
Affinity chromatography of AChE in heparin-agarose The heparin-agarose was prepared as 0.4 ml columns in blue pipette tips, washed with 10 vols. of 10 mM Tris-HCl (pH 7.4), 0.2 M NaCI3. The enzyme was obtained by solubilization of AChE from the endplate region with 10 mM Tris-HCl buffer, 1 M NaCI. After centrifugation, the supernatant was dialyzed against 1000 vols. of 10 mM Tris-HCl (pH 7.4) buffer 0.1 M NaCI, 2 mM EDTA at 4 °C for 12 h. The non-diffusible material was centrifuged 10,000 g for 15 min and the pellet containing the asymmetric AChE forms, was resuspended in the same buffer plus 0.2 M NaCI, and then was layered on the top of the affinity resin. The adsorbed material was washed first with 5 vols. of column buffer, and then was eluted with 2 mg/ml of the different GAGs in 10 mM Tris-HCI (pH 7.4) buffer.
o
AChE assay AChE activity was measured by the method of Ellman et ai. 7. All determinations were done at 37 °C (except for the sedimentation analysis) in a 1-ml reaction mixture containing 100 mM sodium phosphate buffer (pH 7.0), 0.75 mM acetylcholine iodide and 0.3 mM dithio-bt~-nitrobenzoic acid.
1
0
0
........ -
~'- 80 I-(.9 6O
~
< w 4o Q)
< 20
_ ~
,
,
00
0.5
,.
10
[GAGs]
,
15
20
(mg/mt)
Fig. 1. Solubilization of AChE activity from the rat neuromuscular junction of diaphragm, by GAGs. GAGs were added to the Tris-HCl buffer in order to obtain the final concentration shown in the figure. After homogenization, the different aliquots were centrifuged at 10,000 g for 15 min, and the AChE activity was determined in the supernatant. Each point represents the average of 3 different experiments, and AChE activity was determined in triplicate. AChE activity is expressed as percentage of AChE activity solubilized by heparin. Chondroitin sulfate (O), de-sulfated heparin (O), heparan sulfate (A), heparin (El), heparin low molecular weight (1).
Elution of asymmetric AChE from heparin-agarose columns by different GAGs We have previously shown that heparin was able to
Sedimentation analysis To visualize the AChE forms solubilized by the different GAGs, samples of 200 HI of each supernatant were layered onto 5-20% (w/v) sucrose gradients containing 10 mM Tris-HC1 (pH 7.4), 0.5% Triton X-100, 5 mM EDTA, 1 M NaCI and protease inhibitors. To isolate AChE-DS complexes the gradient contains dermatan sulfate 1 mg/ml and does not contain NaCI. Gradients were run at 48,000 rpm at 4 °C for 8 h (4 h for the AChE-dermatan sulfate complexes) in a Beckman SW-65 rotor TM. Thirty to 40 fractions were collected. The AChE activity was assayed by the method of Ellman et al.7 at 4 °C overnight.
•
release the asymmetric A C h E from heparin-agarose column 3, we therefore studied the ability of different G A G s to elute the asymmetric A C h E bound to a heparin-agarose column. For this purpose the columns were loaded with endplate asymmetric forms and t h e y were eluted with 2 mg/ml of different G A G s , Fig. 2 shows the elution pattern obtained with the different G A G s . The results confirm that heparin is by far the best G A G to displace the A C h E b o u n d to the column, other GAGs
have
a weak
effect,
ranging from 6%
for
chondroitin sulfate to 30% for low molecular weight heparin.
RESULTS >-
Solubilization of asymmetric AChE from the rat neuromuscular junction We studied the extraction of A C h E activity by differe n t G A G s in the e n d p l a t e regions of the rat diaphragm muscle. For p u r p o s e of c o m p a r i s o n we normalized the activity solubilized in each case with the activity solubilized by heparin. In the first experiments we compared how m u c h A C h E activity was released by the different G A G s . Fig. 1 shows the esterase activity released by different G A G s . T h e low molecular weight heparin shows a n a p p a r e n t higher capacity to solubilize A C h E t h a n the s t a n d a r d u n f r a c t i o n a t e d heparin normalized as a 100% of A C h E solubilization.
--
30
NoCl
< 2O LU
8
I--- 2C
91
Elsevier BRES 15893
Dermatan sulfate and de-sulfated heparin solubilized collagen-tailed acetylcholinesterase from the rat neuromuscular junction Rommy von Bernhardi* and Nibaldo C. Inestrosa Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences and Medicine, Catholic University of Chile, Santiago (Chile) (Accepted 27 March 1990)
Key words: Aeetyleholinesterase; Asymmetric form; Proteoglycan; Skeletal muscle; Synapsis
We are interested in the study of the interactions involved in the attachment of collagen-tailed acetylcholinesterase (ACHE) to the synaptic basal lamina. The fact that AChE occupies less than 0.1% of the muscle basal lamina, suggests that there is a very high specificity in the interaction that defines its distribution. We have previously found that asymmetric AChE is bound to the neuromuscular junction via heparan sulfate proteoglycans. Sulfated glycosaminoglycans as heparan sulfate and heparin extracted the asymmetric AChE from the synaptic basal lamina. Here we show that dermatan sulfate as well as de-sulfated heparin, are also able to extract collagen-tailed ACHE. Taking into account that the sohibilization of the asymmetric AChE is concomitant with the liberation of a dermatan sulfate proteoglycan from the rat neuromuscular junction, the present results open the possibility that the collagen-tailed AChE is also anchored to dermatan sulfate proteoglycans at the synaptic basal lamina.
INTRODUCTION
A c e t y l c h o l i n e s t e r a s e (ACHE, E C 3.1.1.7) is a polym o r p h i c enzyme which m a y be classified in globular and asymmetric forms 2. T h e asymmetric forms are concent r a t e d at the n e u r o m u s c u l a r junction and consist of 3 t e t r a m e r s linked to a triple helix tail. T h e enzyme is specifically associated to the basal lamina by the collagenlike tail t h r o u g h proteoglycans 4. Glycosaminoglycans ( G A G s ) are the main c o m p o nents of b a s e m e n t m e m b r a n e proteoglycans. F o r their polyanionic character, G A G s can bind o t h e r molecules, especially cationic amines, p e p t i d e s and proteins, some of which are k n o w n to be essential for cell function 9'12. In particular at cholinergic synapses, h e p a r a n sulfate residues a p p e a r to be associated with acetylcholine r e c e p t o r clusters and with the collagen-tailed A C h E 11. L a t e l y it was shown that heparin, besides releasing asymmetric A C h E from the basal lamina of skeletal muscle, specifically solubilizes a d e r m a t a n sulfate proteoglycan ( D S P G ) 6. This result was the first evidence of the existence of D S P G at the skeletal muscle cells surface and suggested a possible association b e t w e e n asymmetric A C h E and D S P G at the muscle basal lamina.
We r e p o r t here that d e r m a t a n sulfate solubilizes asymmetric A C h E from the muscle basal lamina but only detaches a minimal a m o u n t of the A C h E b o u n d to an h e p a r i n - a g a r o s e column. A d d i t i o n a l e x p e r i m e n t s show that de-sulfated heparin as well as low-molecular weight h e p a r i n also release a s y m m e t r i c A C h E from the rat n e u r o m u s c u l a r junction. MATERIALS AND METHODS The following chemicals were used: heparin from bovine lung and porcine intestinal mucosa (including low molecular weight heparin), heparan sulfate, heparin de-sulfated and chondroitin sulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Heparinagarose was purchased from Bethesda Research Laboratories (Gaithersbnrg, MA). Other reagents, where not specified, were obtained from commercial sources.
Surgical procedures Male Sprague-Dawley rats of 250-300 g were maintained with free access to food and water. Their diaphragms were removed from the ribs, washed and kept in ice-cold saline. The muscles were then divided into endplate and non-endplate regionst3. Only the endplate region were used in this work.
AChE extraction procedure The experiments were performed using sequential extraction procedures. The tissue was weighed, finely minced with scissors and homogenized (1:10 w/v) in low ionic strength buffer, 25 mM
* Present address: Department of Biology, University of Utah, Salt Lake City, UT, U.S.A.
Correspondence: N.C. Inestrosa, Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences and Medicine, Catholic University of Chile, P.O. Box l14-D, Santiago, Chile. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
92 Tris-HCI (pH 7.4), 1% Triton X-100 in the presence of protease inhibitors: 2 mM EDTA, 0.1 mM N-ethylmaleimide. 20 Hg/ml benzamidine, 0.25 mg/ml soy-bean trypsin inhibitor and 1 mg/ml bacitracine. Homogenization was performed in a glass-to-glass homogenizer. The homogenate was centrifuged at 10,000 g during 15 min in an Eppendorf centrifuge, and the solubilized AChE activity was determined in the supernatant. All procedures were conducted at 4 °C. Usually 3-4 sequential extractions were carried out, in order to remove most of the globular AChE forms attached to cellular membranes. After centrifugation, the residual pellet was rehomogenized in the same above buffer twice. The last pellet was resuspended (1:5 w/v) in a low ionic strength buffer and then it was separated in various aliquots that were rehomogenized in Tris buffer containing the different GAGs at required concentration. Finally, the GAG-containingsamples were centrifuged at 10,000 g during 15 rain, and the AChE activity was determined in the supernatant.
Affinity chromatography of AChE in heparin-agarose The heparin-agarose was prepared as 0.4 ml columns in blue pipette tips, washed with 10 vols. of 10 mM Tris-HCl (pH 7.4), 0.2 M NaCI3. The enzyme was obtained by solubilization of AChE from the endplate region with 10 mM Tris-HCl buffer, 1 M NaCI. After centrifugation, the supernatant was dialyzed against 1000 vols. of 10 mM Tris-HCl (pH 7.4) buffer 0.1 M NaCI, 2 mM EDTA at 4 °C for 12 h. The non-diffusible material was centrifuged 10,000 g for 15 min and the pellet containing the asymmetric AChE forms, was resuspended in the same buffer plus 0.2 M NaCI, and then was layered on the top of the affinity resin. The adsorbed material was washed first with 5 vols. of column buffer, and then was eluted with 2 mg/ml of the different GAGs in 10 mM Tris-HCI (pH 7.4) buffer.
o
AChE assay AChE activity was measured by the method of Ellman et ai. 7. All determinations were done at 37 °C (except for the sedimentation analysis) in a 1-ml reaction mixture containing 100 mM sodium phosphate buffer (pH 7.0), 0.75 mM acetylcholine iodide and 0.3 mM dithio-bt~-nitrobenzoic acid.
1
0
0
........ -
~'- 80 I-(.9 6O
~
< w 4o Q)
< 20
_ ~
,
,
00
0.5
,.
10
[GAGs]
,
15
20
(mg/mt)
Fig. 1. Solubilization of AChE activity from the rat neuromuscular junction of diaphragm, by GAGs. GAGs were added to the Tris-HCl buffer in order to obtain the final concentration shown in the figure. After homogenization, the different aliquots were centrifuged at 10,000 g for 15 min, and the AChE activity was determined in the supernatant. Each point represents the average of 3 different experiments, and AChE activity was determined in triplicate. AChE activity is expressed as percentage of AChE activity solubilized by heparin. Chondroitin sulfate (O), de-sulfated heparin (O), heparan sulfate (A), heparin (El), heparin low molecular weight (1).
Elution of asymmetric AChE from heparin-agarose columns by different GAGs We have previously shown that heparin was able to
Sedimentation analysis To visualize the AChE forms solubilized by the different GAGs, samples of 200 HI of each supernatant were layered onto 5-20% (w/v) sucrose gradients containing 10 mM Tris-HC1 (pH 7.4), 0.5% Triton X-100, 5 mM EDTA, 1 M NaCI and protease inhibitors. To isolate AChE-DS complexes the gradient contains dermatan sulfate 1 mg/ml and does not contain NaCI. Gradients were run at 48,000 rpm at 4 °C for 8 h (4 h for the AChE-dermatan sulfate complexes) in a Beckman SW-65 rotor TM. Thirty to 40 fractions were collected. The AChE activity was assayed by the method of Ellman et al.7 at 4 °C overnight.
•
release the asymmetric A C h E from heparin-agarose column 3, we therefore studied the ability of different G A G s to elute the asymmetric A C h E bound to a heparin-agarose column. For this purpose the columns were loaded with endplate asymmetric forms and t h e y were eluted with 2 mg/ml of different G A G s , Fig. 2 shows the elution pattern obtained with the different G A G s . The results confirm that heparin is by far the best G A G to displace the A C h E b o u n d to the column, other GAGs
have
a weak
effect,
ranging from 6%
for
chondroitin sulfate to 30% for low molecular weight heparin.
RESULTS >-
Solubilization of asymmetric AChE from the rat neuromuscular junction We studied the extraction of A C h E activity by differe n t G A G s in the e n d p l a t e regions of the rat diaphragm muscle. For p u r p o s e of c o m p a r i s o n we normalized the activity solubilized in each case with the activity solubilized by heparin. In the first experiments we compared how m u c h A C h E activity was released by the different G A G s . Fig. 1 shows the esterase activity released by different G A G s . T h e low molecular weight heparin shows a n a p p a r e n t higher capacity to solubilize A C h E t h a n the s t a n d a r d u n f r a c t i o n a t e d heparin normalized as a 100% of A C h E solubilization.
--
30
NoCl
< 2O LU
8
I--- 2C
