591
Biochem. J. (1990) 266, 591-595 (Printed in Great Britain)
Extracellular accumulation of small dermatan sulphate proteoglycan II by interference with the secretion-recapture pathway Gerd SCHMIDT, Heinz HAUSSER and Hans KRESSE* Institute of Physiological Chemistry and Pathobiochemistry, University of Miinster, Miinster, Federal Republic of Germany
Human skin fibroblasts were metabolically labelled in the presence of affinity-purified antibodies against the core protein of small dermatan sulphate proteoglycan II. The treatment resulted in a dose- and timedependent accumulation of this proteoglycan in the culture medium, with a 2-3-fold increase found within an experimental period of 4 h. The presence of antibodies was without influence on the rate of biosynthesis of the proteoglycan. However, proteoglycan-antibody complexes were inefficiently endocytosed. Addition of unlabelled proteoglycan, which served as a competitor for uptake, similarly led to an accumulation of newly formed [35S]sulphate-labelled proteoglycans. Proteoglycan accumulation also occurred as a consequence of its binding to collagen fibrils which were physically separated from the cell layer. Together, these results establish the quantitative importance of the secretion-recapture pathway of small dermatan sulphate proteoglycan II in cultured fibroblasts.
INTRODUCTION Cultured human skin fibroblasts from juvenile and adult donors synthesize several types of proteoglycans, including a large chondroitin sulphate proteoglycan with a core protein of Mr approx. 400000 (Krusius et al., 1987), a variety of heparan sulphate proteoglycans (Garner & Culp, 1981 ; Woods et al., 1985; Coster et al., 1986) and a small dermatan sulphate proteoglycan with an Mr36319 core protein (Krusius & Ruoslahti, 1986). The last proteoglycan, known as small dermatan sulphate proteoglycan II (DS-PG II) (Heinegard et al., 1985; Rosenberg et al., 1985) or decorin (Yamaguchi & Ruoslahti, 1988), is quantitatively the most important (Gl6ssl et al., 1984). It is predominantly secreted into the culture medium, but it may also associate with cellsurface-bound fibronectin (Schmidt et al., 1987). In the tissue, it binds to the 'd' band of collagen fibrils (Scott & Orford, 1981; Scott & Haigh, 1985), thereby influencing collagen fibrillogenesis (Vogel et al., 1984; Hedbom & Heinegard, 1989). Previously it has been shown that, in cultured fibroblasts, about 30 0 of the newly synthesized proteoglycans are transferred directly to the lysosomal compartment, where complete degradation to monomeric constituents takes place (Fratantoni et al., 1968). For transport of the extracellularly located proteoglycan to the lysosomes, DS-PG II is equipped with a recognition marker on its core protein (Glossl et al., 1983) which interacts with an endocytosis receptor (Hausser et al., 1989). Uptake is remarkably efficient, and a single fibroblast may internalize up to 2 pmol of proteoglycan-bound GlcA/h (Prinz et al., 1978). Nevertheless, the contribution of receptor-mediated endocytosis to the turnover of DS-PG
II in tissue culture has not yet been investigated. We show in this paper that the interference with DS-PG II endocytosis by interaction with core-directed antibodies or by binding to collagen fibrils results in excessive extracellular proteoglycan accumulation.
EXPERIMENTAL Materials A rabbit antiserum against the core protein of DS-PG II from fibroblast secretions was obtained (Glossl et al., 1984) and affinity-purified (Voss et al., 1986) as described previously. Briefly, the antiserum was first passed through a column of Sepharose 4B covalently linked with DS-PG II core protein. Bound antibodies were desorbed with 7 M-urea and, after dialysis, applied to a second affinity matrix containing bovine serum albumin that had been substituted with hexeneuronosyl-GalNAc-4/6-sulphate residues. Antibodies not retarded by this column were monospecific for epitopes on the DS-PG II core protein. Control bovine IgG, Protein A-Sepharose and acidsoluble type I collagen from calf skin were from Sigma, Deisenhofen, Germany, and bovine serum albumin (research grade, cat. no. 11920) was from Serva, Heidelberg, Germany. [35S]Sulphate (carrier-free) and L-[4,53H]leucine (sp. radioactivity 1.8 TBq/mol) were from Amersham-Buchler, Braunschweig, Germany.
Quantification of DS-PG II production Human skin fibroblasts from juvenile or adult human donors were cultivated in modified Eagle's minimum essential medium as described previously (Cantz et al., 1972). At 2-3 days before the experiments, cells were
Abbreviation used: DS-PG II, small dermatan sulphate proteoglycan from fibroblasts. * Correspondence address: Institut fur Physiologische Chemie und Pathobiochemie, Waldeyerstrasse 15, 4400
Germany.
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592
treated with trypsin and plated to obtain confluency within the next 48 h. For studies on the quantification of DS-PG II production, the medium was changed to Waymouth MAB 87/3 medium, whose composition was as listed in the Gibco catalogue except that MgSO4 was replaced with an equimolar amount of MgCl2, and bovine serum albumin was added to a final concentration of 10 g/litre. Upon incubation of fibroblasts in a 25 cm2 Falcon plastic flask with [35S]sulphate, medium (2 ml) was mixed with proteinase inhibitors (Gl6ssl et al., 1984). In experiments where antibodies were present in the culture medium during the incubation period, the proteoglycan-antibody complexes were purified by adsorption to Protein A-Sepharose. The complexes were allowed to react with 5 mg of Protein A-Sepharose by end-over-end rotation for about 15 h at 5 'C. After centrifugation, the sediment was washed with 3 x I ml of 20 mM-Tris/HCl buffer, pH 7.4, containing 1 M-NaCl, 0. 500 deoxycholate, 0.500 Triton X- 100 and proteinase inhibitors (buffer A), and then with 2 x 1 ml ofphosphatebuffered saline (137 mM-NaCl/2.7 mM-KCl/16.1 mMNa2HPO4/ 1.9 mM-KH2PO4, pH 7.4). The proteoglycan was then solubilized by boiling with 1 % SDS. The supernatant containing unbound DS-PG II was mixed with an equal volume of double-concentrated buffer A and treated with Protein A-Sepharose that had been preadsorbed with antiserum (Gl6ssl et al., 1984). Cellassociated immunoreactive material was obtained in the same way after extraction with detergents (Gl6ssl et al., 1984). In the case of competition experiments with unlabelled DS-PG II, [35S]sulphate incorporation into proteoglycans was quantified by chromatography on DEAE-Trisarcryl (Gl6ssl et al., 1984). Parallel incubations without unlabelled DS-PG II showed that [35S]sulphate incorporation could be quantified with a coefficient of variation of 2 0 (n = 6). Other methods Preparation of unlabelled DS-PG II from fibroblast secretions (Gl6ssl et al., 1984) and quantification of endocytosis of exogenously added proteoglycans (Gl6ssl et al., 1983) were performed exactly as described. Collagen fibrils were obtained by incubating a mixture of 20 mg of collagen in 6 ml of 16.7 mM-acetic acid and 12 ml of phosphate-buffered saline, pH 7.4, for 1 h at 37 IC, followed by centrifugation. The pellet was suspended in albumin-free Waymouth medium, dispersed by ultrasonication and distributed after resuspension in albumin-containing Waymouth medium into 30 mm Millicell-HA culture plate inserts (Millipore, Eschborn, Germany) that were placed on top of cultures in 35 mm x 14 mm multiwell plates (Costar, Cambridge, MA, U.S.A.). Uronic acids were quantified according to Bitter & Muir (1962).
RESULTS AND DISCUSSION Accumulation of DS-PG II in the presence of antibodies [35S]Sulphate incorporation into DS-PG II was measured in monolayer cultures in the presence of affinity-purified antibodies. Antibody-bound proteoglycans were quantified by addition of Protein ASepharose; unbound DS-PG II was subsequently isolated by its reactivity with Protein A-Sepharose which had been pre-adsorbed with antiserum. It is shown in Fig. I that the addition of increasing doses of DS-PG II
G. Schmidt, H. Hausser and H. Kresse
12
10 E
8 0L C')
x
4-
20
0.23
1.15
0.23
5.75
IgG
1.15
5.75
(,ug/ml)
Fig. 1. Antibody-dependence of DS-PG II accumulation Confluent fibroblast cultures in 25 cm2 flasks were incubated with 2 ml of Waymouth medium which contained 10 g of bovine serum albumin/litre, the IgG concentrations indicated, and 180 kBq of [35S]sulphate. Streptomycin sesquisulphate was omitted. After 4 h of incubation, media were analysed for antibody-bound DS-PG 11 (-) and total DS-PG II (a). Bovine IgG was used in the control incubations.
antibodies led to the accumulation of increasing amounts of DS-PG II in the culture medium. Control immunoglobulins were ineffective. On studying the time course of proteoglycan production, it was found that in the presence of DS-PG II antibodies there was a much greater increase with time of the amount of the proteoglycan compared with cultures treated with control IgG. No significant differences were seen between antibodytreated and control cultures in the amount of cellassociated DS-PG II (Fig. 2). Also, there was no increase in other proteoglycan types which can be found in the supernatant remaining after immune precipitation. The accumulation of DS-PG II in the culture medium could be the result either of increased biosynthesis or of reduced endocytosis. The biosynthesis of DS-PG II was measured by pulse-labelling of DS-PG II with either [3H]leucine or [35S]sulphate after preincubation with the respective antibodies. Table 1 shows that pretreatment with antibodies did not result in a significant alteration in the biosynthesis of DS-PG II core protein or glycosaminoglycan chains. There was also no significant increase in the polysaccharide chain length, since the electrophoretic mobility of the proteoglycan remained unaltered (results not shown). However, the rate of endocytosis of an immune complex of [35S]sulphatelabelled DS-PG II was much lower than the uptake rate of proteoglycan treated with control IgG (Table 2). This inefficient clearance of IgG-DS-PG II complexes occurred despite the fact that skin fibroblasts are equipped with a receptor for aggregated IgG (Frey et al., 1984). 1990
Accumulation of small proteoglycan
593 0
[35S]Sulphate-labelled proteoglycan (50000 c.p.m.) and 200 ,ul of fetal calf serum were mixed with Hepes-buffered culture medium and 100 ,ul of antiserum or control serum to give a final volume of 2.0 ml. The solutions were dialysed for 16 h at 5 °C against 200 ml of serum-free endocytosis medium before DS-PG II uptake was measured over a period of 6 h.
4E 6
Table 2. Endocytosis of antibody-bound DS-PG II
3-
CD)
Antiserum
Control serum
26
56
19
19
=6
0-
x
2-
Clearance (/ulh per mg of protein) Degradation (% of endocytosed amount) Binding to cell membrane (% of added amount)
0~~~~~
-
1
0~~~~~~~ 0
3.6
3.0
0
0
12
34
Time (h)
Fig. 2. Time-dependence of DS-PG II accumulation Fibroblasts were challenged as described in the legend to Fig. 1 with a concentration of 11.5,ug/flask of affinitypurified antibodies against DS-PG II core protein (-, A) or of control IgG (0, A) for the times indicated. *, 0, Sum of cell-associated and secreted DS-PG II; A, A, cellassociated DS-PG II.
Together, these results suggest that, within an experimental period of 4 h, at least half or even more of the newly synthesized DS-PG II is endocytosed in monolayer fibroblast cultures. These experiments do not however Table 1. Incorporation of 13Hlleucine and 135Slsulphate into DSPG II after pretreatment with antibodies
Confluent fibroblast cultures in 25 cm2 flasks were incubated for 3.5 h with 2 ml of leucine-free Waymouth medium containing 10 g of bovine serum albumin/litre instead of fetal calf serum, and either 1l.5 ,ug of affinitypurified antibodies against DS-PG II core protein or control IgG. [3H]Leucine (2.2 MBq) was then added, and the incubation was continued for an additional 30 min. DS-PG II core protein was then quantified in the cell layer as described in the Experimental section. Radioactive DSPG II was not detectable in the culture medium. In a separate experiment performed in the same way, leucinecontaining medium was used, and 3.7 MBq of [35S]sulphate was present during the last 15 min of incubation. About 10% of labelled DS-PG II was found in the culture medium. 103 X [3H]Leucine incorporation
10-3 X [35 S]Sulphate
incorporation
(c.p.m.)
Pretreatment
(c.p.m.)
Affinitypurified IgG Control IgG
14.7
24.7
14.1
23.2
Vol. 266
indicate whether only secreted proteoglycans are recaptured or whether the endocytosed proteoglycans remain bound to the plasma membrane before they become internalized in the absence of an extracellular ligand. In another set of experiments, antibodies and fibroblasts were physically separated from each other by placing immobilized antibodies in a Millicell culture plate insert on top of the cells, the bottom of the insert being diffusible for the proteoglycan (Table 3). It was again observed that antibody treatment resulted in an increased extracellular DS-PG II concentration. This suggests that the proteoglycan is released from the cell membrane before it becomes internalized. However, it is not yet known why the liquid medium, too, was enriched with proteoglycan. We observed, however, that a certain amount of rabbit IgG was released from the complex with Protein A-Sepharose. Accumulation of I35Slsulphate-labelied DS-PG II in the presence of unlabelled DS-PG and of type I collagen The quantitative importance of endocytosis of DS-PG II was supported by experiments in which the amount of secreted [35S]sulphate-labelled DS-PG II was quantified upon exposure of fibroblasts to an increasing dose of unlabelled DS-PG II, which should serve as a competitive inhibitor for uptake of labelled proteoglycan (Table 4). As predicted, there was an increase in the amount of radioactive proteoglycan recovered from the medium with increasing concentrations of unlabelled DS-PG II. Assuming that only 85 % of secreted proteoglycans were represented by DS-PG II (Rauch et al., 1986), a comparison of the amounts of extracellular [35S]sulphatelabelled DS-PG II found in the absence and in the presence of the highest dose of unlabelled competitor indicated that 24 % of DS-PG II secreted in the absence of the competitive inhibitor had been endocytosed. This value represents a minimal estimate, since the uptake of [35S]sulphate-labelled DS-PG II might not be abolished at the highest inhibitor concentration. An iterative estimation, using the Dixon equation (Dixon, 1953), suggested that at the highest inhibitor concentration, uptake was still 5 % of the secreted amount. Thus about 30 % of secreted DS-PG II should have been endocytosed in this experiment in the absence of unlabelled proteoglycan.
594
G. Schmidt, H. Hausser and H. Kresse
Table 3. Antibody-dependence of DS-PG II accumulation
Confluent fibroblast cultures in 35 mm x 14 mm multiwell plates were exposed to Protein A-Sepharose coated with IgG. The gel was placed into 30 mm Millicell-HA culture plate inserts. Incubation in 2 ml of medium was for 4 h at 37 °C on a rocking platform in the presence of 0.5 MBq of [35S]sulphate. 10-3 x [35S]DS-PG II (c.p.m.)
Conditions ... Localization of DS-PG II
Antibody-coated
Amount of IgG ...
Protein A-bound Liquid medium Cell layer
Control IgG-coated
Protein A-Sepharose
Protein A-Sepharose
18 mg
4.5 mg
18 mg
4.5 mg
1.2 2.7 1.0
0.8 2.6 1.1
None 1.7 0.9
None 1.8 1.1
Finally, type I collagen fibrils placed into Millicell culture plate inserts were used instead of antibodies as DS-PG II-binding agents. Immobilization of DS-PG II on collagen fibrils also led to a severalfold increase in extracellular accumulation of the proteoglycan (Table 5). Taken together, the results reported in this paper provide evidence that DS-PG II undergoes a secretionrecapture pathway in cultured fibroblasts. There are, however, problems with the quantitative interpretation of the data. The rates of endocytosis of DS-PG II measured as clearance of exogenously supplied proteoglycan varied between about 20 and 100 ,ll/h per mg of cell protein (Prinz et al., 1978; Gl6ssl et al., 1983). These clearance rates do not imply that interference with endocytosis should lead to a severalfold extracellular accumulation of the proteoglycan within 4 h. On the other hand, it had been recognized that the core protein, which has to interact with the endocytosis receptor (Gl6ssl et al., 1983), is very sensitive to denaturing conditions, e.g. dialysis against water, lyophilization, etc. It seems possible therefore that the clearance rates measured under seemingly optimal conditions are still artifactually too low. An exact calculation of the fraction of DS-PG II endocytosed in the absence or the presence of antibodies is also hampered by the fact that the antibody-DS-PG II complexes are also subject to
receptor-mediated endocytosis. Without this, the portion of extracellular DS-PG II accumulating as antibody complexes would be even greater. The quantitative importance of endocytosis in the metabolism of DS-PG II in fibroblasts raises the question of the physiological role of the secretion-recapture pathway in vivo. DS-PG II could escape from this pathway by binding to collagen fibrils (Table 5). Endocytosis of DS-PG II could therefore be a means of adjusting the amount of extracellular proteoglycan to the proteoglycan-binding capacity of extracellular matrix constituents. However, more physiological models than monolayer cultures are required for an investigation of the regulation of the extracellular DS-PG II concentration. Regardless of the uncertainties in the quantitative interpretation of the data, our results indicate that, in monolayer cultures, the incorporation of radioactive precursors into extracellular DS-PG II is reflected by the difference between proteoglycan secretion and recapture even in short-term experiments. Any conclusions on the modulation of DS-PG II synthesis by exogenously supplied agents or by variations in the tissue culture con-
Table 4. Accumulation of 135Slsulphate-labelled DS-PG II in the presence of unlabelled DS-PG II
Confluent fibroblast cultures in 35 mm x 14 mm multiwell plates were exposed to type I collagen fibrils which had been placed into 30 mm Millicell-HA culture plate inserts. Inserts without collagen were put in control cultures. Incubation in 4 ml of medium was for 19 h at 37 °C on a rocking platform in the presence of 50 kBq of [35S]sulphate. Collagen-bound radioactivity was solubilized, after extensive washing, by boiling in 400 ,ul of 1 % SDS.
Confluent fibroblast cultures 4 days after subculturing were incubated for 4 h with I ml of medium containing 0.5 MBq of [35S]sulphate and the indicated amount of unlabelled DS-PG II.
Table 5. Accumulation of I35Slsulphate-labelled DS-PG II in the presence of type I collagen fibrils
10-3 x [35S]Sulphate incorporation
10-3 x [35S]Sulphate incorporation (c.p.m.)
(c.p.m.) Unlabelled DS-PG II (nmol of GlcA) 0 2 12 57
Fraction ...
Culture medium
layer
Cell
15.1 15.6 17.1 18.8
7.4 7.2 7.8 7.0
Collagen added (mg) None 3 6 9
Fraction ...
Collagen- Liquid bound medium 6.9 8.0 9.5
1.4 0.7 0.6 0.7
Cell
layer 0.6 0.7 0.7 0.5
1990
595
Accumulation of small proteoglycan
ditions are therefore valid only if the influence of the manipulation on the endocytosis of the proteoglycan has been taken into consideration. We are indebted to Petra Blumberg for her skilled technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 310, Teilprojekt B2).
REFERENCES Bitter, T. & Muir, H. (1962) Anal. Biochem. 4, 330-334 Cantz, M., Kresse, H., Barton, R. W. & Neufeld, E. F. (1972) Methods Enzymol. 28, 884-897 C6ster, L., Carlstedt, I., Kendall, S., Malmstr6m, A., Schmidtchen, A. & Fransson, L.-A. (1986) J. Biol. Chem. 261, 12079-12088 Dixon, M. (1953) Biochem. J. 55, 170-171 Fratantoni, J. C., Hall, C. W. & Neufeld, E. F. (1968) Proc. Natl. Acad. Sci. U.S.A. 60, 699-706 Frey, J., Quentin, H.-J. & Afting, E. G. (1984) Eur. J. Immunol. 14, 1115-1118 Garner, J. A. & Culp, L. A. (1981) Biochemistry 20, 7350-7359 Gl6ssl, J., Schubert-Prinz, R., Gregory, J. D., Damle, S. P., von Figura, K. & Kresse, H. (1983) Biochem. J. 215, 295-301 Gl6ssl, J., Beck, M. & Kresse, H. (1984) J. Biol. Chem. 259, 14144-14150 Hausser, H., Hoppe, W., Rauch, U. & Kresse, H. (1989) Biochem. J. 263, 137-142 Received 4 September 1989/31 October 1989; accepted 16 November 1989
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Hedbom, E. & Heinegatrd, D. (1989) J. Biol. Chem. 264, 6898-6905 Heinegard, D., Bj6rne-Persson, A., C6ster, L., Franzen, A., Gardell, S., Malmstr6m, A., Paulsson, M., Sandfalk, R. & Vogel, K. (1985) Biochem. J. 230, 181-194 Krusius, T. & Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7683-7687 Krusius, T., Gehlsen, K. R. & Ruoslahti, E. (1987) J. Biol. Chem. 262, 13120-13125 Prinz, R., Schwermann, J., Buddecke, E. & von Figura, K. (1978) Biochem. J. 176, 671-676 Rauch, U., Gl6ssl, J. & Kresse, H. (1986) Biochem. J. 238, 465-474 Rosenberg, L. C., Choi, H. U., Tang, L.-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A. & Poole, A. R. (1985) J. Biol.
Chem. 260, 6304-6313 Schmidt, G., Robenek, H., Harrach, H., Gl6ssl, J., Nolte, V., Hormann, H., Richter, H. & Kresse, H. (1987) J. Cell Biol. 104, 1683-1691 Scott, J. E. & Haigh, M. (1985) Biosci. Rep. 5, 71-82 Scott, J. E. & Orford, C. R. (1981) Biochem. J. 197, 213-216 Vogel, K. G., Paulsson, M. & Heinegatrd, D. (1984) Biochem. J. 223, 587-597 Voss, B., Gl6ssl, J., Cully, Z. & Kresse, H. (1986) J. Histochem. Cytochem. 34, 1013-1019 Woods, A., Couchman, J. R. & Ho6k, M. (1985) J. Biol. Chem. 260, 10872-10879 Yamaguchi, Y. & Ruoslahti, E. (1988) Nature (London) 336, 244-246
Biochem. J. (1990) 266, 591-595 (Printed in Great Britain)
Extracellular accumulation of small dermatan sulphate proteoglycan II by interference with the secretion-recapture pathway Gerd SCHMIDT, Heinz HAUSSER and Hans KRESSE* Institute of Physiological Chemistry and Pathobiochemistry, University of Miinster, Miinster, Federal Republic of Germany
Human skin fibroblasts were metabolically labelled in the presence of affinity-purified antibodies against the core protein of small dermatan sulphate proteoglycan II. The treatment resulted in a dose- and timedependent accumulation of this proteoglycan in the culture medium, with a 2-3-fold increase found within an experimental period of 4 h. The presence of antibodies was without influence on the rate of biosynthesis of the proteoglycan. However, proteoglycan-antibody complexes were inefficiently endocytosed. Addition of unlabelled proteoglycan, which served as a competitor for uptake, similarly led to an accumulation of newly formed [35S]sulphate-labelled proteoglycans. Proteoglycan accumulation also occurred as a consequence of its binding to collagen fibrils which were physically separated from the cell layer. Together, these results establish the quantitative importance of the secretion-recapture pathway of small dermatan sulphate proteoglycan II in cultured fibroblasts.
INTRODUCTION Cultured human skin fibroblasts from juvenile and adult donors synthesize several types of proteoglycans, including a large chondroitin sulphate proteoglycan with a core protein of Mr approx. 400000 (Krusius et al., 1987), a variety of heparan sulphate proteoglycans (Garner & Culp, 1981 ; Woods et al., 1985; Coster et al., 1986) and a small dermatan sulphate proteoglycan with an Mr36319 core protein (Krusius & Ruoslahti, 1986). The last proteoglycan, known as small dermatan sulphate proteoglycan II (DS-PG II) (Heinegard et al., 1985; Rosenberg et al., 1985) or decorin (Yamaguchi & Ruoslahti, 1988), is quantitatively the most important (Gl6ssl et al., 1984). It is predominantly secreted into the culture medium, but it may also associate with cellsurface-bound fibronectin (Schmidt et al., 1987). In the tissue, it binds to the 'd' band of collagen fibrils (Scott & Orford, 1981; Scott & Haigh, 1985), thereby influencing collagen fibrillogenesis (Vogel et al., 1984; Hedbom & Heinegard, 1989). Previously it has been shown that, in cultured fibroblasts, about 30 0 of the newly synthesized proteoglycans are transferred directly to the lysosomal compartment, where complete degradation to monomeric constituents takes place (Fratantoni et al., 1968). For transport of the extracellularly located proteoglycan to the lysosomes, DS-PG II is equipped with a recognition marker on its core protein (Glossl et al., 1983) which interacts with an endocytosis receptor (Hausser et al., 1989). Uptake is remarkably efficient, and a single fibroblast may internalize up to 2 pmol of proteoglycan-bound GlcA/h (Prinz et al., 1978). Nevertheless, the contribution of receptor-mediated endocytosis to the turnover of DS-PG
II in tissue culture has not yet been investigated. We show in this paper that the interference with DS-PG II endocytosis by interaction with core-directed antibodies or by binding to collagen fibrils results in excessive extracellular proteoglycan accumulation.
EXPERIMENTAL Materials A rabbit antiserum against the core protein of DS-PG II from fibroblast secretions was obtained (Glossl et al., 1984) and affinity-purified (Voss et al., 1986) as described previously. Briefly, the antiserum was first passed through a column of Sepharose 4B covalently linked with DS-PG II core protein. Bound antibodies were desorbed with 7 M-urea and, after dialysis, applied to a second affinity matrix containing bovine serum albumin that had been substituted with hexeneuronosyl-GalNAc-4/6-sulphate residues. Antibodies not retarded by this column were monospecific for epitopes on the DS-PG II core protein. Control bovine IgG, Protein A-Sepharose and acidsoluble type I collagen from calf skin were from Sigma, Deisenhofen, Germany, and bovine serum albumin (research grade, cat. no. 11920) was from Serva, Heidelberg, Germany. [35S]Sulphate (carrier-free) and L-[4,53H]leucine (sp. radioactivity 1.8 TBq/mol) were from Amersham-Buchler, Braunschweig, Germany.
Quantification of DS-PG II production Human skin fibroblasts from juvenile or adult human donors were cultivated in modified Eagle's minimum essential medium as described previously (Cantz et al., 1972). At 2-3 days before the experiments, cells were
Abbreviation used: DS-PG II, small dermatan sulphate proteoglycan from fibroblasts. * Correspondence address: Institut fur Physiologische Chemie und Pathobiochemie, Waldeyerstrasse 15, 4400
Germany.
Vol. 266
Miinster, Federal Republic of
592
treated with trypsin and plated to obtain confluency within the next 48 h. For studies on the quantification of DS-PG II production, the medium was changed to Waymouth MAB 87/3 medium, whose composition was as listed in the Gibco catalogue except that MgSO4 was replaced with an equimolar amount of MgCl2, and bovine serum albumin was added to a final concentration of 10 g/litre. Upon incubation of fibroblasts in a 25 cm2 Falcon plastic flask with [35S]sulphate, medium (2 ml) was mixed with proteinase inhibitors (Gl6ssl et al., 1984). In experiments where antibodies were present in the culture medium during the incubation period, the proteoglycan-antibody complexes were purified by adsorption to Protein A-Sepharose. The complexes were allowed to react with 5 mg of Protein A-Sepharose by end-over-end rotation for about 15 h at 5 'C. After centrifugation, the sediment was washed with 3 x I ml of 20 mM-Tris/HCl buffer, pH 7.4, containing 1 M-NaCl, 0. 500 deoxycholate, 0.500 Triton X- 100 and proteinase inhibitors (buffer A), and then with 2 x 1 ml ofphosphatebuffered saline (137 mM-NaCl/2.7 mM-KCl/16.1 mMNa2HPO4/ 1.9 mM-KH2PO4, pH 7.4). The proteoglycan was then solubilized by boiling with 1 % SDS. The supernatant containing unbound DS-PG II was mixed with an equal volume of double-concentrated buffer A and treated with Protein A-Sepharose that had been preadsorbed with antiserum (Gl6ssl et al., 1984). Cellassociated immunoreactive material was obtained in the same way after extraction with detergents (Gl6ssl et al., 1984). In the case of competition experiments with unlabelled DS-PG II, [35S]sulphate incorporation into proteoglycans was quantified by chromatography on DEAE-Trisarcryl (Gl6ssl et al., 1984). Parallel incubations without unlabelled DS-PG II showed that [35S]sulphate incorporation could be quantified with a coefficient of variation of 2 0 (n = 6). Other methods Preparation of unlabelled DS-PG II from fibroblast secretions (Gl6ssl et al., 1984) and quantification of endocytosis of exogenously added proteoglycans (Gl6ssl et al., 1983) were performed exactly as described. Collagen fibrils were obtained by incubating a mixture of 20 mg of collagen in 6 ml of 16.7 mM-acetic acid and 12 ml of phosphate-buffered saline, pH 7.4, for 1 h at 37 IC, followed by centrifugation. The pellet was suspended in albumin-free Waymouth medium, dispersed by ultrasonication and distributed after resuspension in albumin-containing Waymouth medium into 30 mm Millicell-HA culture plate inserts (Millipore, Eschborn, Germany) that were placed on top of cultures in 35 mm x 14 mm multiwell plates (Costar, Cambridge, MA, U.S.A.). Uronic acids were quantified according to Bitter & Muir (1962).
RESULTS AND DISCUSSION Accumulation of DS-PG II in the presence of antibodies [35S]Sulphate incorporation into DS-PG II was measured in monolayer cultures in the presence of affinity-purified antibodies. Antibody-bound proteoglycans were quantified by addition of Protein ASepharose; unbound DS-PG II was subsequently isolated by its reactivity with Protein A-Sepharose which had been pre-adsorbed with antiserum. It is shown in Fig. I that the addition of increasing doses of DS-PG II
G. Schmidt, H. Hausser and H. Kresse
12
10 E
8 0L C')
x
4-
20
0.23
1.15
0.23
5.75
IgG
1.15
5.75
(,ug/ml)
Fig. 1. Antibody-dependence of DS-PG II accumulation Confluent fibroblast cultures in 25 cm2 flasks were incubated with 2 ml of Waymouth medium which contained 10 g of bovine serum albumin/litre, the IgG concentrations indicated, and 180 kBq of [35S]sulphate. Streptomycin sesquisulphate was omitted. After 4 h of incubation, media were analysed for antibody-bound DS-PG 11 (-) and total DS-PG II (a). Bovine IgG was used in the control incubations.
antibodies led to the accumulation of increasing amounts of DS-PG II in the culture medium. Control immunoglobulins were ineffective. On studying the time course of proteoglycan production, it was found that in the presence of DS-PG II antibodies there was a much greater increase with time of the amount of the proteoglycan compared with cultures treated with control IgG. No significant differences were seen between antibodytreated and control cultures in the amount of cellassociated DS-PG II (Fig. 2). Also, there was no increase in other proteoglycan types which can be found in the supernatant remaining after immune precipitation. The accumulation of DS-PG II in the culture medium could be the result either of increased biosynthesis or of reduced endocytosis. The biosynthesis of DS-PG II was measured by pulse-labelling of DS-PG II with either [3H]leucine or [35S]sulphate after preincubation with the respective antibodies. Table 1 shows that pretreatment with antibodies did not result in a significant alteration in the biosynthesis of DS-PG II core protein or glycosaminoglycan chains. There was also no significant increase in the polysaccharide chain length, since the electrophoretic mobility of the proteoglycan remained unaltered (results not shown). However, the rate of endocytosis of an immune complex of [35S]sulphatelabelled DS-PG II was much lower than the uptake rate of proteoglycan treated with control IgG (Table 2). This inefficient clearance of IgG-DS-PG II complexes occurred despite the fact that skin fibroblasts are equipped with a receptor for aggregated IgG (Frey et al., 1984). 1990
Accumulation of small proteoglycan
593 0
[35S]Sulphate-labelled proteoglycan (50000 c.p.m.) and 200 ,ul of fetal calf serum were mixed with Hepes-buffered culture medium and 100 ,ul of antiserum or control serum to give a final volume of 2.0 ml. The solutions were dialysed for 16 h at 5 °C against 200 ml of serum-free endocytosis medium before DS-PG II uptake was measured over a period of 6 h.
4E 6
Table 2. Endocytosis of antibody-bound DS-PG II
3-
CD)
Antiserum
Control serum
26
56
19
19
=6
0-
x
2-
Clearance (/ulh per mg of protein) Degradation (% of endocytosed amount) Binding to cell membrane (% of added amount)
0~~~~~
-
1
0~~~~~~~ 0
3.6
3.0
0
0
12
34
Time (h)
Fig. 2. Time-dependence of DS-PG II accumulation Fibroblasts were challenged as described in the legend to Fig. 1 with a concentration of 11.5,ug/flask of affinitypurified antibodies against DS-PG II core protein (-, A) or of control IgG (0, A) for the times indicated. *, 0, Sum of cell-associated and secreted DS-PG II; A, A, cellassociated DS-PG II.
Together, these results suggest that, within an experimental period of 4 h, at least half or even more of the newly synthesized DS-PG II is endocytosed in monolayer fibroblast cultures. These experiments do not however Table 1. Incorporation of 13Hlleucine and 135Slsulphate into DSPG II after pretreatment with antibodies
Confluent fibroblast cultures in 25 cm2 flasks were incubated for 3.5 h with 2 ml of leucine-free Waymouth medium containing 10 g of bovine serum albumin/litre instead of fetal calf serum, and either 1l.5 ,ug of affinitypurified antibodies against DS-PG II core protein or control IgG. [3H]Leucine (2.2 MBq) was then added, and the incubation was continued for an additional 30 min. DS-PG II core protein was then quantified in the cell layer as described in the Experimental section. Radioactive DSPG II was not detectable in the culture medium. In a separate experiment performed in the same way, leucinecontaining medium was used, and 3.7 MBq of [35S]sulphate was present during the last 15 min of incubation. About 10% of labelled DS-PG II was found in the culture medium. 103 X [3H]Leucine incorporation
10-3 X [35 S]Sulphate
incorporation
(c.p.m.)
Pretreatment
(c.p.m.)
Affinitypurified IgG Control IgG
14.7
24.7
14.1
23.2
Vol. 266
indicate whether only secreted proteoglycans are recaptured or whether the endocytosed proteoglycans remain bound to the plasma membrane before they become internalized in the absence of an extracellular ligand. In another set of experiments, antibodies and fibroblasts were physically separated from each other by placing immobilized antibodies in a Millicell culture plate insert on top of the cells, the bottom of the insert being diffusible for the proteoglycan (Table 3). It was again observed that antibody treatment resulted in an increased extracellular DS-PG II concentration. This suggests that the proteoglycan is released from the cell membrane before it becomes internalized. However, it is not yet known why the liquid medium, too, was enriched with proteoglycan. We observed, however, that a certain amount of rabbit IgG was released from the complex with Protein A-Sepharose. Accumulation of I35Slsulphate-labelied DS-PG II in the presence of unlabelled DS-PG and of type I collagen The quantitative importance of endocytosis of DS-PG II was supported by experiments in which the amount of secreted [35S]sulphate-labelled DS-PG II was quantified upon exposure of fibroblasts to an increasing dose of unlabelled DS-PG II, which should serve as a competitive inhibitor for uptake of labelled proteoglycan (Table 4). As predicted, there was an increase in the amount of radioactive proteoglycan recovered from the medium with increasing concentrations of unlabelled DS-PG II. Assuming that only 85 % of secreted proteoglycans were represented by DS-PG II (Rauch et al., 1986), a comparison of the amounts of extracellular [35S]sulphatelabelled DS-PG II found in the absence and in the presence of the highest dose of unlabelled competitor indicated that 24 % of DS-PG II secreted in the absence of the competitive inhibitor had been endocytosed. This value represents a minimal estimate, since the uptake of [35S]sulphate-labelled DS-PG II might not be abolished at the highest inhibitor concentration. An iterative estimation, using the Dixon equation (Dixon, 1953), suggested that at the highest inhibitor concentration, uptake was still 5 % of the secreted amount. Thus about 30 % of secreted DS-PG II should have been endocytosed in this experiment in the absence of unlabelled proteoglycan.
594
G. Schmidt, H. Hausser and H. Kresse
Table 3. Antibody-dependence of DS-PG II accumulation
Confluent fibroblast cultures in 35 mm x 14 mm multiwell plates were exposed to Protein A-Sepharose coated with IgG. The gel was placed into 30 mm Millicell-HA culture plate inserts. Incubation in 2 ml of medium was for 4 h at 37 °C on a rocking platform in the presence of 0.5 MBq of [35S]sulphate. 10-3 x [35S]DS-PG II (c.p.m.)
Conditions ... Localization of DS-PG II
Antibody-coated
Amount of IgG ...
Protein A-bound Liquid medium Cell layer
Control IgG-coated
Protein A-Sepharose
Protein A-Sepharose
18 mg
4.5 mg
18 mg
4.5 mg
1.2 2.7 1.0
0.8 2.6 1.1
None 1.7 0.9
None 1.8 1.1
Finally, type I collagen fibrils placed into Millicell culture plate inserts were used instead of antibodies as DS-PG II-binding agents. Immobilization of DS-PG II on collagen fibrils also led to a severalfold increase in extracellular accumulation of the proteoglycan (Table 5). Taken together, the results reported in this paper provide evidence that DS-PG II undergoes a secretionrecapture pathway in cultured fibroblasts. There are, however, problems with the quantitative interpretation of the data. The rates of endocytosis of DS-PG II measured as clearance of exogenously supplied proteoglycan varied between about 20 and 100 ,ll/h per mg of cell protein (Prinz et al., 1978; Gl6ssl et al., 1983). These clearance rates do not imply that interference with endocytosis should lead to a severalfold extracellular accumulation of the proteoglycan within 4 h. On the other hand, it had been recognized that the core protein, which has to interact with the endocytosis receptor (Gl6ssl et al., 1983), is very sensitive to denaturing conditions, e.g. dialysis against water, lyophilization, etc. It seems possible therefore that the clearance rates measured under seemingly optimal conditions are still artifactually too low. An exact calculation of the fraction of DS-PG II endocytosed in the absence or the presence of antibodies is also hampered by the fact that the antibody-DS-PG II complexes are also subject to
receptor-mediated endocytosis. Without this, the portion of extracellular DS-PG II accumulating as antibody complexes would be even greater. The quantitative importance of endocytosis in the metabolism of DS-PG II in fibroblasts raises the question of the physiological role of the secretion-recapture pathway in vivo. DS-PG II could escape from this pathway by binding to collagen fibrils (Table 5). Endocytosis of DS-PG II could therefore be a means of adjusting the amount of extracellular proteoglycan to the proteoglycan-binding capacity of extracellular matrix constituents. However, more physiological models than monolayer cultures are required for an investigation of the regulation of the extracellular DS-PG II concentration. Regardless of the uncertainties in the quantitative interpretation of the data, our results indicate that, in monolayer cultures, the incorporation of radioactive precursors into extracellular DS-PG II is reflected by the difference between proteoglycan secretion and recapture even in short-term experiments. Any conclusions on the modulation of DS-PG II synthesis by exogenously supplied agents or by variations in the tissue culture con-
Table 4. Accumulation of 135Slsulphate-labelled DS-PG II in the presence of unlabelled DS-PG II
Confluent fibroblast cultures in 35 mm x 14 mm multiwell plates were exposed to type I collagen fibrils which had been placed into 30 mm Millicell-HA culture plate inserts. Inserts without collagen were put in control cultures. Incubation in 4 ml of medium was for 19 h at 37 °C on a rocking platform in the presence of 50 kBq of [35S]sulphate. Collagen-bound radioactivity was solubilized, after extensive washing, by boiling in 400 ,ul of 1 % SDS.
Confluent fibroblast cultures 4 days after subculturing were incubated for 4 h with I ml of medium containing 0.5 MBq of [35S]sulphate and the indicated amount of unlabelled DS-PG II.
Table 5. Accumulation of I35Slsulphate-labelled DS-PG II in the presence of type I collagen fibrils
10-3 x [35S]Sulphate incorporation
10-3 x [35S]Sulphate incorporation (c.p.m.)
(c.p.m.) Unlabelled DS-PG II (nmol of GlcA) 0 2 12 57
Fraction ...
Culture medium
layer
Cell
15.1 15.6 17.1 18.8
7.4 7.2 7.8 7.0
Collagen added (mg) None 3 6 9
Fraction ...
Collagen- Liquid bound medium 6.9 8.0 9.5
1.4 0.7 0.6 0.7
Cell
layer 0.6 0.7 0.7 0.5
1990
595
Accumulation of small proteoglycan
ditions are therefore valid only if the influence of the manipulation on the endocytosis of the proteoglycan has been taken into consideration. We are indebted to Petra Blumberg for her skilled technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 310, Teilprojekt B2).
REFERENCES Bitter, T. & Muir, H. (1962) Anal. Biochem. 4, 330-334 Cantz, M., Kresse, H., Barton, R. W. & Neufeld, E. F. (1972) Methods Enzymol. 28, 884-897 C6ster, L., Carlstedt, I., Kendall, S., Malmstr6m, A., Schmidtchen, A. & Fransson, L.-A. (1986) J. Biol. Chem. 261, 12079-12088 Dixon, M. (1953) Biochem. J. 55, 170-171 Fratantoni, J. C., Hall, C. W. & Neufeld, E. F. (1968) Proc. Natl. Acad. Sci. U.S.A. 60, 699-706 Frey, J., Quentin, H.-J. & Afting, E. G. (1984) Eur. J. Immunol. 14, 1115-1118 Garner, J. A. & Culp, L. A. (1981) Biochemistry 20, 7350-7359 Gl6ssl, J., Schubert-Prinz, R., Gregory, J. D., Damle, S. P., von Figura, K. & Kresse, H. (1983) Biochem. J. 215, 295-301 Gl6ssl, J., Beck, M. & Kresse, H. (1984) J. Biol. Chem. 259, 14144-14150 Hausser, H., Hoppe, W., Rauch, U. & Kresse, H. (1989) Biochem. J. 263, 137-142 Received 4 September 1989/31 October 1989; accepted 16 November 1989
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Hedbom, E. & Heinegatrd, D. (1989) J. Biol. Chem. 264, 6898-6905 Heinegard, D., Bj6rne-Persson, A., C6ster, L., Franzen, A., Gardell, S., Malmstr6m, A., Paulsson, M., Sandfalk, R. & Vogel, K. (1985) Biochem. J. 230, 181-194 Krusius, T. & Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7683-7687 Krusius, T., Gehlsen, K. R. & Ruoslahti, E. (1987) J. Biol. Chem. 262, 13120-13125 Prinz, R., Schwermann, J., Buddecke, E. & von Figura, K. (1978) Biochem. J. 176, 671-676 Rauch, U., Gl6ssl, J. & Kresse, H. (1986) Biochem. J. 238, 465-474 Rosenberg, L. C., Choi, H. U., Tang, L.-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A. & Poole, A. R. (1985) J. Biol.
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