Comp. Biochem. PhysioL Vol. 100B,No. 3, pp. 565-570, 1991 Printed in Great Britain
0305-0491/91 $3.00+ 0.00 © 1991PergamonPresspie
ISOLATION A N D CHARACTERIZATION OF RAT SKELETAL MUSCLE PROTEOGLYCAN DECORIN A N D COMPARISON WITH THE H U M A N FIBROBLAST DECORIN WINSTONANDRADEand ENRIQUEBRANDAN* Molecular Neurobiology Unit, Department of Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, P.O. Box l l4-D, Santiago, Chile (Fax: 56 2 222 5515) (Received 23 April 1991)
Abstract--l. The extracellular matrix (ECM) of rat skeletal muscle contains several proteoglycans (PGs). The more abundant correspond to a chondroitin/dermatan sulfate PG or decorin. 2. Decorin isolated from rat skeletal muscle ECM has a smaller molecular size than human fibroblast decorin. 3. The difference in size is mainly due to the glycosaminoglycan (GAG) chain length rather than the core protein size. 4. Peptide analysis of trypsin treated decorins shows at least three peptides with the same electrophoretic mobility.
(Brandan and Inestrosa, 1984; Brandan et al., 1985). Immunocytolocalization experiments have shown that acetylcholine receptor aggregates are associated with plaques of basement membrane beparan sulfate PGs on the surface of the skeletal muscle (Anderson and Fambrough, 1984). We have recently shown that denervation of adult rat leg muscles caused an increase in the synthesis of PGs present at the ECM and that the level of expression of a particular chondroitin/dermatan sulfate PG (DSPG-II), similar or identical to decorin appears to be regulated by motor nerve activity (Fadic et al., 1990; Brandan et al., 1990). This particular PG is widely distributed in the ECM of skin (G16ssl et al., 1984), tendon (Vogel and Evanko, 1987), bone (Fisher et al., 1983), cartilage (Rosenberg et al., 1985) and a variety of other connective tissues, being the fibroblast-like cells the principal cell type responsible for their synthesis. The cDNA for human decodn has been sequenced corresponding to a tool. wt of 36,319 (Krusius and Ruoslahti, 1986). The PG decorin seems to be crucial for the organization of ECM because the core protein presents the ability to bind collagen and fibronectin (Vogel et al., 1984; Lewandowska et al., 1987; Schmidt et al., 1987). In this report we have biochemically compared decorin synthesized by rat skeletal muscle with the well-characterized decorin synthesized by human fibroblasts.
Skeletal muscle fibers are surrounded by a specialized form of extracellular matrix (ECM), the basal lamina. This structure is highly specialized at the neuromuscular junction region (Covault and Sanes, 1985; Sanes et al., 1986), being implicated in the nerve-muscle interaction and the muscle regeneration (Sanes et al., 1978). Special interest has been focused on understanding the components of the basal lamina. The isolation and characterization of extracelhilar matrix components are, therefore, one prerequisite in order to ascertain their functional roles. Proteoglycans (PGs) are widely distributed in the ECM of all mammalian tissues (Heinegard and Paulsson, 1984; Fransson, 1987; Ruoslahti, 1988) as well as in association with the plasma membrane of eukaryotic cells (H66k et al., 1984; Brandan and Hirschberg, 1989). Several PGs have been isolated from skeletal muscle ECM. Thus, three major highbuoyant-density chondroitin sulfate PGs have been isolated from embryonic chick skeletal muscle (Carrino and Caplan, 1982, 1984); A low tool. wt chondroitin sulfate PG has been isolated from rabbit skeletal muscle (Parthasarathy and Tanzer, 1987); and two heparan sulfate PGs (Brandan and Inestrosa, 1987a) and a dermatan sulfate PG, displaced by the glycosaminoglycan (GAG) heparin have been isolated from rat skeletal muscle (Brandan and Inestrosa, 1987b). Several observations suggest that PGs present at the muscle basal lamina might play important functional roles. Thus, a heparan sulfate PG is concentrated at the neuromuscular junction (Bayne et al., 1984), and a related PG is responsible for the anchoring of the asymmetric form of acetylcholinesterase to the synaptic basal lamina
MATERIALSAND METHODS Materials The following materials were purchased from the supplier indicated: Na23SSO4 carrier free was obtained from New England Nuclear, Boston, MA, USA. Na ~2sIfrom Chilean Nuclear Commission, Santiago, Chile; chondroitinase ABC lyase, chondroitinase AC, benzamidine hydrochloride, DEAE-Sephacel, Sepharose CL-6B from Sigma Chemical
*Author to whom correspondence should be addressed. 565
WINSTON ANDRADE and ENRIQUEBRANDAN
Co., St. Louis, MO, USA. Heparitinase from Miles Laboratories, USA. Other reagents were obtained from commercial sources. Methods Male Sprague-Dawley rats (200-250 g) were injected intraperitonally with 1.5-2.0mCi of [35S]-sulfate in saline. Leg muscles were removed 18 hr after the injection (Fadic et al., 1990). Isolation o f rat muscle proteoglycans Leg muscles were removed, homogenized in 10mM Tris-HCl buffer, pH 7.4, containing 5 mM benzamidine, 50mM 6-aminohexanoic acid, 10mM N-ethylmaleimide, 10 mM EDTA and 0.5% (v/v) Triton X-100 and centrifuged in a Sorval SS-34 rotor at 12,000g for 15 min at 4°C. The pellet was resuspended in the same homogenation buffer and centrifuged again. This last step was repeated twice. Supernatants were pooled and the labeled-PGs, present in the final pellet, were extracted as previously described (Brandan and Inestrosa, 1987a). The detergent insoluble pellet was solubilized with 6 M guanidine-HC1 and 50 mM sodium acetate, pH 5.8, containing protease inhibitors, for 12 hr at 4°C with continuous agitation. The extract was centrifuged as above and unincorporated radioactive precursors, guanidine-HCl and salts were removed by dialysis against 100 volumes of 4 M urea, 0.1 M NaC1, 0.1% Triton X-100, protease inhibitors and 50 mM sodium acetate, pH 5.8, for 6 hr and then against 100 volumes of the same solution but containing 8 M urea, for another 6 h r at 4°C. After dialysis the radioactive material was applied to a DEAE-Sephacel column pre-equilibrated in the same 8 M urea buffer as previously described (Yanagishita and Hascall, 1984). The column was washed with 8 ml of the same buffer and then eluted with a continuous NaC1 gradient, 0.1-1.0 M NaC1 in the same buffer (60 ml total volume). Elution was performed at a flow rate of 5.0 ml/hr and fractions of 1.0 ml were collected. The NaC1 gradient was monitored by measuring conductivity of the fractions. The labeled eluted material was pooled, dialyzed for 12 hr against 10mM Tris-HC1 buffer, pH 7.4, 0.5% Triton X-100 and 0.1 M NaC1 containing protease inhibitors and precipitated by the addition of 2.5 volumes of cold ethanol. The labeled-PGs were collected by centrifugation and resuspended in 0.5 ml of 10mM Tris-HC1 buffer, pH 7.4, and 0.1 M NaC1. Human decorin was purified from secretion media of human fibroblast, IMR-90 (Nichols et al., 1976), exactly as described previously (Gl6ssl et al., 1984). Filtration chromatography Pooled fractions from the DEAE-Sephacel column were fractionated on a Sepharose CL-6B column equilibrated with 1% sodium dodecyl sulfate (SDS), 0.1 M NaC1 and 50mM Tris-HC1 buffer, pH8.0. Samples (0.5ml) were applied to the column together with previously fractionated blue Dextran (2000) and phenol red, to mark void and total volumes, respectively. Columns were run at a flow rate of 5.0 ml/hr and effluent fractions of 1.0 ml were collected, counted for radioactivity and pooled before cold ethanol precipitation. Sulfated material which eluted in fractions corresponding to a K~v of 0.50, which is mainly rat skeletal muscle decorin (Fadic et al., 1990), were pooled, concentrated and treated to obtain the GAGs chains as explained below. The GAGs chains were fractionated under the same conditions as above. Enzymatic treatments and chemical analyses Chondroitinase ABC and AC treatments of labeled-PGs were done exactly as previously described (Brandan and Inestrosa, 1987a). Trypsin treatment of the core proteins was done as follows. Purified decorin from rat skeletal muscle and human fibroblast was iodinated, treated with chondroitinase ABC followed by fractionation in
SDS-PAGE and the core proteins excised from the gel. The iodinated core proteins were incubated with trypsin 5 #g/ml at 4°C for 15 min. After the treatment the samples were separated in a 13.5% SDS-PAGE, followed by an autoradiography. GAGSs chains were removed from the PGs exactly as described previously by us (Fadic et al., 1990). lodination and S D S - P A G E analysis o f proteoglycans Purified PGs after Sepharose CL-6B chromatography were iodinated using the chloramine T method (Carpenter and Cohen, 1976). Sulfated samples and iodinated PGs were analyzed by electrophoresis on I0 and 13.5% SDS-PAGE and fluorographed as described previously (Carlson and Wright, 1987). RESULTS Table 1 shows the c o m p o s i t i o n of different P G s f o u n d in rat muscle E C M a n d h u m a n fibroblast secretion. A l t h o u g h the P G sources are quite different some similarities in the overall c o m p o s i t i o n can be observed. A t least three different PGs, evaluated by sensitivity to G A G lyases are found. In each case the more a b u n d a n t P G corresponds to a c h o n d r o i t i n / d e r m a t a n sulfate PG. This P G c o r r e s p o n d s to decorin a n d is present in several E C M of different tissues. We were interested in characterizing the small c h o n d r o i t i n / d e r m a t a n sulfate P G present in rat skeletal muscle described by us ( B r a n d a n et al., 1990) with the one secreted by h u m a n fibroblast (G16ssl et al., 1984). To do this, decorin was purified from rat skeletal muscle E C M a n d culture media containing secreted P G s from h u m a n fibroblasts using a combin a t i o n o f c h r o m a t o g r a p h y on D E A E - S e p h a c e l a n d gel fractionation (Sepharose CL-6B). The purified P G s were separated in 10% S D S - P A G E followed by fluorography. Figure 1, indicates t h a t the rat muscle decorin has a mol. wt of 80,000-100,000 (lane 1) while h u m a n decorin has a higher mol. wt (95,000-120,000, lane 3). As expected b o t h sulfated P G s were totally degraded after t r e a t m e n t with c h o n d r o i t i n a s e A B C (Fig. 1, lanes 2 a n d 4). To confirm the difference in size o f b o t h c h o n d r o i t i n a s e A B C sensitive PGS, they were fractionated o n a Sepharose CL-6B. Figure 2(A) indicates t h a t h u m a n decorin eluted with a Kay o f 0.35 where rat muscle decorin eluted with a Kay of 0.45. To evaluate if the observed difference in size was due to the length o f the G A G chains attached to the core proteins, G A G s chains were isolated from each P G u n d e r the conditions described in Materials a n d M e t h o d s , a n d c h r o m a t o g r a p h e d o n a Sepharose Table 1. Proteoglycans composition of rat skeletal muscle extracellular matrix and secretion of human fibroblast in culture Muscle* Fibroblastt Type of proteoglycan (%) (%) Chondroitin sulfate 6 13 Chondroitin/dermatan sulfate 54 67 Heparan sulfate 23 20 Others 16 0 The values correspond to an average of two independent determinations. *DEAE-Sephacel bound PGs were eluted with 1.0 M NaCI and treated with chondroitinase ABC, AC, heparitinase and nitrous acid. The reaction products were fractionated on Sepharose CL-6B as explained in Materials and Methods. "['Human fibroblast secreted PGs were concentrated on a DEAE-Sephacel column and treated as above.
Comparison between rat skeletal muscle and human fibroblast decorin
1 16 -
8 4 _
Fig. 1. Rat skeletal muscle ECM contain a chondroitin/dermatan sulfate proteoglycan smaller in size than decorin synthesized by human fibroblast. 3~S-Sulfated decorin was purified from rat skeletal muscle ECM and culture media of human fibroblast as described under Materials and Methods. An aliquot containing 20,000 cpm of sulfated proteoglycan was incubated with chondroitinase ABC before electrophoresis on a 10% SDS-PAGE. Control; lanes 1 and 3. Chondroitinase ABC treatment, lanes 2 and 4. The mol. wts for several standards are indicated in each case. CL-6B. Figure 2(B) clearly indicates a difference in the size of the G A G s . Larger G A G chains were isolated from human decorin (K~v of 0.5) compared to rat muscle decorin (K~v of 0.62). These results indicate that the difference in size observed between both PGs is probably due to the G A G s length of the chains present in each PG. The next step was to determine the molecular size of the core protein present in each decorin. To do this, the purified PGs were iodinated and treated with
chondroitinase ABC and the core proteins separated in a 13.5% S D S - P A G E gel. Figure 3, lanes 1 and 4, show the ionidated core proteins for rat muscle and human fibroblast, respectively. In the case of the rat muscle a 37,000 core protein was found, while for human fibroblast a core protein of 39,000 was observed. It is already known that differences in the core sequence can exist between decorin from different sources (Day et al., 1987). In order to compare both core proteins, the protein cores were digested with
o;o 0;2 o;~ 0;6 o;e KAv
o;~ 0;2 o;~ 0;6 o'.e fo K,o,v
Fig. 2. Dccorin isolated from rat skeletal muscle has smaller glycosaminoglycan chains than human fibroblast dccorin. (A) Intact decorin isolated from rat skeletal muscle ECM (closed circles) and human fibroblast (open circles) was fractionated on Scpharosc CL-6B as explained in Materials and Methods. (B) GAGs chains were removed by alkali in the presence of sodium borohydride from each proteoglycan and fractionated in the same column. The recoveries in both columns were in the range 89-97%.
Fig. 3. Both core proteins generated some similar peptides after trypsin treatment. The purified decorins were iodinated and the core proteins obtained after chondroitinase ABC treatment purified as described in Materials and Methods. Lanes I and 4 correspond to the core protein obtained from rat skeletal muscle decorin and human fibroblast decorin, respectively. Each sample was treated with trypsin before separation on a 13.5% SDS-PAGE. Lanes 2 and 3 correspond to the trypsin treated samples.
trypsin under mild conditions as described in Materials and Methods, and the peptides generated by the treatment separated and compared in the same gel. Figure 3, lanes 2 and 3, indicate that trypsin generated some peptides which produced an identical electrophoretic mobility (Fig. 3, indicated by double arrows); however some peptides with different mobility were also observed. The mol. wts of the identical generated peptides were 17,000, 12,000 and 8000, respectively. However, small peptides of 26,000 and 7000 were only observed in the rat muscle decorin. These results revealed some similarity between both core protein isolated from human and rat tissue. DISCUSSION
In this paper we have isolated, characterized and compared the structural characteristics of decorin synthesized by rat skeletal muscle with human fibroblast decorin. Both PGs have the same similarities and differences. Thus both are totally sensitive to
chondroitinase ABC, while only 30% sensitive to chondroitinase AC (data not shown). The hydrodynamic characteristics as well as the mol. wt determined by SDS-PAGE indicate that rat skeletal muscle decorin has a lower mol. wt compared to human fibroblast decorin. This difference seems to be mainly due to a difference in the size of the GAGs; the chain length of the G A G s obtained from rat muscle decorin is smaller than the one obtained from human fibroblast decorin. This was shown by the different elution position observed after Sepharose CL-6B chromatography for the respective isolated GAGs. A high Kay of 0.62 for rat muscle decorin G A G s compared to a Kav of 0.45 for the G A G s obtained from human fibroblast decorin was observed. However, only a small difference in the mol. wt of the core protein was observed after iodination of the respective decorin was followed by chondroitinase ABC treatment. A mol. wt of 37,000 for rat decorin was given compared to 39,000 for the human decorin. This difference can be attributed to
Comparison between rat skeletal muscle and human fibroblast decorin dissimilarity in the protein length and/or different post-translational modifications. It is known that human decorin contains several N-linked carbohydrates (Glfssl et al., 1984) and is also phosphorylated (G16ssl et aL, 1986). The exact mol. wt for the cloned human decorin is 36,319 (Krusius and Ruoslahti, 1986), a value quite similar to the one found for both decorins. The trypsin treatment of both core proteins generated several peptides that presented an identical electrophoretic mobility. Three peptides of identical electrophoretic mobility were found. The presence of resistant peptides of 17,000 and 12,000 were described previously for human fibroblast decorins after treatment with a staphylococcal protease (V8) (Glrssl et al., 1984). The fact that three of the five peptides obtained after trypsin treatment presented identical mol. wt indicate that both decorins are rather similar in structure. However, the presence of two different peptides might be a result of different amino acidic sequences and/or different degrees of posttranslational modifications such as glycosilation. We have immunoprecipitated rat muscle decorin using polyclonal antibodies against rat fibroblast decorin (Brandan et aL, 1990). However the antibodies did not precipitate the human decorin (data not shown). This PG seems to be a very important component of skeletal muscle ECM. For instance, it can be specifically solubilized by GAGs such as heparin (Brandan and Inestrosa, 1987b) and the level of expression of muscle decorin is clearly influenced by the muscle activity, because after muscle denervation or paralysis of the muscle there is a three-fold increase in their expression (Fadic et al., 1990; Brandan et al., 1990). Another interesting observation, is the fact that during in vitro differentiation of mouse skeletal cells, there is an important increase in the synthesis of this PG (Brandan et al., 1991). Decorin has the ability to bind collagen and fibronectin through a specific binding domain present in its protein structure (Ruoslahti, 1989). We have shown that in rat skeletal muscle, decorin is concentrated at ECM located either at the perimisium or the endomisium (Brandan et al., 1990). The exact functional role(s) of this PG in the skeletal muscle ECM remains to be elucidated, but the fact that the level of expression is influenced either by the presence of the nerve and during the cell differentiation (Fadic et al., 1990; Brandan et al., 1991) makes this PG a very attractive component of the skeletal muscle ECM. REFERENCES Anderson M. J. and Fambrough D. M. (1984) Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fbers. J. Cell Biol. 97, 1396-1411. Bayne E. K., Anderson M. J. and Fambrough M. D. (1984) Extracellular matrix organization in developing muscle: Correlation with acetylcholineaggregates. J. Cell Biol. 99, 1486-1501. Brandan E. and Inestrosa N. C. (1984) Binding of the asymmetric forms of acetylcholinesteraseto heparin. Biochem. J. 221, 415-422. Brandan E., Maldonado M., Garrido J. and Inestrosa N. C. (1985) Anchorage of collagen-tailed acetylcholinesterase
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