567th MEETING, DURHAM
443
present predominantly as sialic acid and glucosamine (either free or N-acetylated). Thus it appears that the epimerization step is a major event in the utilization of N-acetylgalactosamine in the isolated perfused rat liver. A. D. M. gratefully acknowledges receipt of a grant from the M.R.C.
Curtis, C. G., Powell, G. M. & Stone, S. L. (1970) J. Physiol. (London) 213, 14 Kay, J. (1976) Int. J. Pept. Protein Res. 8, 379-384 Kraan, J. G. & Muir, H. (1957) Biochem. J. 66, 5 5 ~ Morley, F., Tarentino, A. L., McGarrahan, J. F. & Delgiacco, R. (1938) Biochem. J. 107, 637-644 Wood, K. M., Wusteman, F. S. & Curtis, C. G. (1973) Biochern. J. 134, 1009-1013
The Degradation of Proteoglycan by Leucocyte Elastase PETER J. ROUGHLEY Strangeways Research Laboratory, Wort’s Causeway, Cambridge CBl 4RN, U.K.
It has now been established that the proteoglycan core protein consists of an hyaluronic acid-binding region and a glycosaminoglycan-attachmentregion (HeinegBrd & Hascall, 1974~).The former is globular in structure and devoid of glycosaminoglycan chains, whereas the latter has an extended structure to which the chondroitin sulphate and keratan sulphate chains are bound (Hardingham et al., 1976). Heineglrd & Hascall (19746) have shown that a sequential digestion of proteoglycan with trypsin and chymotrypsin released chondroitin sulphate clusters containing between one and eight chains, and that about 50% of the keratan sulphate was associated with these clusters. Keiser et al. (1976) have also shown that digestion with elastase from neutrophil leucocytes produced clusters that contained between one and five chondroitin sulphate chains. Leucocyte neutral proteinases may have a prominent role in the degradation of the cartilage extracellular matrix in inflammatory situations. The work presented in the present paper examines the mode of action of leucocyte elastase on cartilage proteoglycan with the aim of answering two questions. First, at what stage in the degradation are the keratan sulphate chains eliminated from the chondroitin sulphate clusters, and secondly, at what stage is the ability to interact with hyaluronic acid lost?
Table 1. Degradation of proteoglycan by elastase Values for percentage decrease in specific viscosity (in parentheses) were calculated relative to proteoglycan (0%) and the final degradation product (100 %). Hexosamine ratios were measured on samples precipitated by the addition of cetylpyridiniurn chloride. For details of experimental procedure, see the text. Specific viscosity (% decrease) 0.57 (0) 0.50 (17) 0.41 (38) 0.36 (50) 0.31 (62) 0.26 (74) 0.21 (86) 0.17 (95) 0.15 (100)
* With more enzyme. Vol. 5
Degradation time (h) 0 0.05 0.15 0.2 0.4 0.7 2 19
44*
GalN/GlcN molar ratio 12.1 12.4 12.5 13.0 13.0 13.4 14.2 16.4 22.5
444
BIOCHEMICAL SOCIETY TRANSACTIONS
by Proteoglycan was extracted from bovine nasal cartilage with ~ M - C ~asCdescribed I~ Roughley & Mason (1976). Purification from protein and hyaluronic acid was by the CsC1-density-gradient procedures of Sajdera & Hascall (1969). The proteoglycan was digested with elastase (EC 3.4.21.11) that had been isolated from human spleen by Dr. P. M. Starkey (Starkey & Barrett, 1976). Digestion mixtures contained proteoglycan (2mg/ml) and elastase (O.lSpg/ml) in 0.2~-Tris/Hcl,pH7.5 at 40°C. Degradation was followed by viscometry, and products were isolated at various times. Isolation of the glycosaminoglycan-peptide fragments was by the addition of 2 5 0 ~ of 1 5 % (w/v) cetylpyridinium chloride to l m l of the digestion mixture, and elastase activity was inhibited by the addition of 20,d of lOOm~-phenylmethanesulphonylfluoride in propan-2-01. By this procedure glycosaminoglycan-peptides that contain only keratan sulphate remain in solution, and so the product that is isolated consists of chondroitin sulphate-containing peptides and mixed chondroitin sulphate/keratan sulphatecontaining peptides (Heinegbrd & Hascall, 19746). The precipitates were washed four times with 1% (w/v) cetylpyridinium chloride, then dissolved in aq. 60% (v/v) propan-1 -01. After reprecipitation by the addition of ethanol saturated with potassium acetate, the precipitate was washed sequentially with ethanol and diethyl ether before drying at 20°C. Galactosamine/glucosaminemolar ratios were determined on a Locarte amino acid analyser by the use of a single buffer system (pH5.28 citrate, as described by the manufacturer). Hydrolysis was at 100°C for 8 h in ~M-H CI, and 5Opg of the precipitated degradation products was used. The results are summarized in Table 1 , together with the specific viscosity of the digestion mixture from which the product was isolated, and the time after addition of the enzyme. The decrease in viscosity was initially very rapid, but slowed progressivelyduring the incubation. In contrast, the removal of keratan sulphate from the chondroitin sulphate-containing peptides was most pronounced in the final stages of degradation, when changes in viscosity were slowest. After incubation for 2 h the viscosity had decreased by 86% of the maximum observed decrease, but only 15 % of the keratan sulphate contained in the proteoglycan had been eliminated. The limiting degradation product, obtained when further addition of elastase produced no further decrease in viscosity, contained 54% of the keratan sulphate found in the proteoglycan. The composition of the degradation products was investigated by electrophoresis in agarose/polyacrylamide gels (McDevitt & Muir, 1971) under the conditions described by Roughley & Mason (1976). In this system the proteoglycan appeared as a broad area of diffuse Toluidine Blue staining with two regions of maximal intensity at Rx 0.8 and 0.9 (relative to Bromophenol Blue). The intermediate degradation products also showed diffuse staining, the average mobility of which increased as the viscosity of the digestion mixture from which they were obtained decreased. In contrast, the limit degradation product appeared as a well-defined band of Rx 1.2. The limit degradation product was not observed until the final stages of degradation, when changes in viscosity were minimal but changes in the hexosamine ratio were maximal. The electrophoretic system was also used to study the interaction of hyaluronic acid with the degradation products. Hyaluronic acid has an Rx value of 0.5, but is not stained under the conditions used. It would be expected that when proteoglycan or a glycosaminoglycan-peptide interacts with hyaluronic acid, staining would be observed. If the interaction is stable to the conditions of electrophoresis staining would be at Rx 0.5, whereas if the interaction is labile, staining would be intermediate between the position of hyaluronic acid and that normally observed for the degradation product. Electrophoresis was performed with a mixture containing equal amounts (w/w) of hyaluronic acid and degradation product. The proteoglycan showed a broad area of diffuse staining from the position of hyaluronic acid to that of the proteoglycan. A similar effect was observed for the initial degradation products, though the degree of retention decreased as the spec& viscosity of the product decreased. No interaction with hyaluronic acid could be detected with the limit degradation product or intermediate products after the viscosity had decreased by 62% of the maximum observed decrease. 1977
567th MEETING, DURHAM
445
The ability of the hyaluronic acid-binding terminus to resist proteolytic attack may be due to its globular conformation. It was concluded that elastase degradation of proteoglycan proceeded by the following pathway. The initial cleavages were within the glycosaminoglycan-attachmentregion and resulted in a large decrease in hydrodynamic size, but there was little release of free keratan sulphate-peptides, and the hyaluronic acid-binding terminus remained functional. Further degradation resulted in the loss of interaction with hyaluronic acid and the formation of small chondroitin sulphate/keratan sulphate-containing clusters. The final stage of degradation involved the loss of keratan sulphate-peptides from the small clusters with little subsequent decrease in their hydrodynamic size. In comparison with the proteoglycan, the clusters of limiting size were relatively homogeneous and appear to be a common feature of all the proteoglycan molecules. Similar electrophoretic patterns have been observed for the intermediate degradation products produced by other tissue proteinases, cathepsins D, B and G, though the size and hexosamine composition of the limit degradation product differed in each case. Thus it could be postulated that the initial cleavages in proteoglycan degradation are common to all proteinases, and that differences occur only in the final cleavages which produce the clusters of limiting size. I thank the Nuffield Foundation for financial support.
Hardingham, T. E., Ewins, R. J. F. & Muir, H. (1976) Bioclzem. J. 157, 127-143 Heinegard, D. & Hascall, V. C. (1974~)J . Biol. Chem. 249,4250-4256 Heinegard, D. & Hascall, V. C. (19746) Arch. Biochern. Biophys. 1 6 5 , 4 2 7 4 1 Keiser, H., Greenwald, R. A., Feinstein, G. & Janoff, A. (1976)J. Clin. Invest. 57,625-632 McDevitt, C . A. & Muir, H. (1971) Anal. Biochem. 44,612-622 Roughley, P. J. & Mason, R. M. (1976) Biochem. J. 157, 357-367 Sajdera, S. W. &. Hascall, V. C. (1969) J. Biol. Chem. 244, 77-87 Starkey, P. M. & Barrett, A. J. (1976) Biochem. J . 155, 255-263
Vol. 5
443
present predominantly as sialic acid and glucosamine (either free or N-acetylated). Thus it appears that the epimerization step is a major event in the utilization of N-acetylgalactosamine in the isolated perfused rat liver. A. D. M. gratefully acknowledges receipt of a grant from the M.R.C.
Curtis, C. G., Powell, G. M. & Stone, S. L. (1970) J. Physiol. (London) 213, 14 Kay, J. (1976) Int. J. Pept. Protein Res. 8, 379-384 Kraan, J. G. & Muir, H. (1957) Biochem. J. 66, 5 5 ~ Morley, F., Tarentino, A. L., McGarrahan, J. F. & Delgiacco, R. (1938) Biochem. J. 107, 637-644 Wood, K. M., Wusteman, F. S. & Curtis, C. G. (1973) Biochern. J. 134, 1009-1013
The Degradation of Proteoglycan by Leucocyte Elastase PETER J. ROUGHLEY Strangeways Research Laboratory, Wort’s Causeway, Cambridge CBl 4RN, U.K.
It has now been established that the proteoglycan core protein consists of an hyaluronic acid-binding region and a glycosaminoglycan-attachmentregion (HeinegBrd & Hascall, 1974~).The former is globular in structure and devoid of glycosaminoglycan chains, whereas the latter has an extended structure to which the chondroitin sulphate and keratan sulphate chains are bound (Hardingham et al., 1976). Heineglrd & Hascall (19746) have shown that a sequential digestion of proteoglycan with trypsin and chymotrypsin released chondroitin sulphate clusters containing between one and eight chains, and that about 50% of the keratan sulphate was associated with these clusters. Keiser et al. (1976) have also shown that digestion with elastase from neutrophil leucocytes produced clusters that contained between one and five chondroitin sulphate chains. Leucocyte neutral proteinases may have a prominent role in the degradation of the cartilage extracellular matrix in inflammatory situations. The work presented in the present paper examines the mode of action of leucocyte elastase on cartilage proteoglycan with the aim of answering two questions. First, at what stage in the degradation are the keratan sulphate chains eliminated from the chondroitin sulphate clusters, and secondly, at what stage is the ability to interact with hyaluronic acid lost?
Table 1. Degradation of proteoglycan by elastase Values for percentage decrease in specific viscosity (in parentheses) were calculated relative to proteoglycan (0%) and the final degradation product (100 %). Hexosamine ratios were measured on samples precipitated by the addition of cetylpyridiniurn chloride. For details of experimental procedure, see the text. Specific viscosity (% decrease) 0.57 (0) 0.50 (17) 0.41 (38) 0.36 (50) 0.31 (62) 0.26 (74) 0.21 (86) 0.17 (95) 0.15 (100)
* With more enzyme. Vol. 5
Degradation time (h) 0 0.05 0.15 0.2 0.4 0.7 2 19
44*
GalN/GlcN molar ratio 12.1 12.4 12.5 13.0 13.0 13.4 14.2 16.4 22.5
444
BIOCHEMICAL SOCIETY TRANSACTIONS
by Proteoglycan was extracted from bovine nasal cartilage with ~ M - C ~asCdescribed I~ Roughley & Mason (1976). Purification from protein and hyaluronic acid was by the CsC1-density-gradient procedures of Sajdera & Hascall (1969). The proteoglycan was digested with elastase (EC 3.4.21.11) that had been isolated from human spleen by Dr. P. M. Starkey (Starkey & Barrett, 1976). Digestion mixtures contained proteoglycan (2mg/ml) and elastase (O.lSpg/ml) in 0.2~-Tris/Hcl,pH7.5 at 40°C. Degradation was followed by viscometry, and products were isolated at various times. Isolation of the glycosaminoglycan-peptide fragments was by the addition of 2 5 0 ~ of 1 5 % (w/v) cetylpyridinium chloride to l m l of the digestion mixture, and elastase activity was inhibited by the addition of 20,d of lOOm~-phenylmethanesulphonylfluoride in propan-2-01. By this procedure glycosaminoglycan-peptides that contain only keratan sulphate remain in solution, and so the product that is isolated consists of chondroitin sulphate-containing peptides and mixed chondroitin sulphate/keratan sulphatecontaining peptides (Heinegbrd & Hascall, 19746). The precipitates were washed four times with 1% (w/v) cetylpyridinium chloride, then dissolved in aq. 60% (v/v) propan-1 -01. After reprecipitation by the addition of ethanol saturated with potassium acetate, the precipitate was washed sequentially with ethanol and diethyl ether before drying at 20°C. Galactosamine/glucosaminemolar ratios were determined on a Locarte amino acid analyser by the use of a single buffer system (pH5.28 citrate, as described by the manufacturer). Hydrolysis was at 100°C for 8 h in ~M-H CI, and 5Opg of the precipitated degradation products was used. The results are summarized in Table 1 , together with the specific viscosity of the digestion mixture from which the product was isolated, and the time after addition of the enzyme. The decrease in viscosity was initially very rapid, but slowed progressivelyduring the incubation. In contrast, the removal of keratan sulphate from the chondroitin sulphate-containing peptides was most pronounced in the final stages of degradation, when changes in viscosity were slowest. After incubation for 2 h the viscosity had decreased by 86% of the maximum observed decrease, but only 15 % of the keratan sulphate contained in the proteoglycan had been eliminated. The limiting degradation product, obtained when further addition of elastase produced no further decrease in viscosity, contained 54% of the keratan sulphate found in the proteoglycan. The composition of the degradation products was investigated by electrophoresis in agarose/polyacrylamide gels (McDevitt & Muir, 1971) under the conditions described by Roughley & Mason (1976). In this system the proteoglycan appeared as a broad area of diffuse Toluidine Blue staining with two regions of maximal intensity at Rx 0.8 and 0.9 (relative to Bromophenol Blue). The intermediate degradation products also showed diffuse staining, the average mobility of which increased as the viscosity of the digestion mixture from which they were obtained decreased. In contrast, the limit degradation product appeared as a well-defined band of Rx 1.2. The limit degradation product was not observed until the final stages of degradation, when changes in viscosity were minimal but changes in the hexosamine ratio were maximal. The electrophoretic system was also used to study the interaction of hyaluronic acid with the degradation products. Hyaluronic acid has an Rx value of 0.5, but is not stained under the conditions used. It would be expected that when proteoglycan or a glycosaminoglycan-peptide interacts with hyaluronic acid, staining would be observed. If the interaction is stable to the conditions of electrophoresis staining would be at Rx 0.5, whereas if the interaction is labile, staining would be intermediate between the position of hyaluronic acid and that normally observed for the degradation product. Electrophoresis was performed with a mixture containing equal amounts (w/w) of hyaluronic acid and degradation product. The proteoglycan showed a broad area of diffuse staining from the position of hyaluronic acid to that of the proteoglycan. A similar effect was observed for the initial degradation products, though the degree of retention decreased as the spec& viscosity of the product decreased. No interaction with hyaluronic acid could be detected with the limit degradation product or intermediate products after the viscosity had decreased by 62% of the maximum observed decrease. 1977
567th MEETING, DURHAM
445
The ability of the hyaluronic acid-binding terminus to resist proteolytic attack may be due to its globular conformation. It was concluded that elastase degradation of proteoglycan proceeded by the following pathway. The initial cleavages were within the glycosaminoglycan-attachmentregion and resulted in a large decrease in hydrodynamic size, but there was little release of free keratan sulphate-peptides, and the hyaluronic acid-binding terminus remained functional. Further degradation resulted in the loss of interaction with hyaluronic acid and the formation of small chondroitin sulphate/keratan sulphate-containing clusters. The final stage of degradation involved the loss of keratan sulphate-peptides from the small clusters with little subsequent decrease in their hydrodynamic size. In comparison with the proteoglycan, the clusters of limiting size were relatively homogeneous and appear to be a common feature of all the proteoglycan molecules. Similar electrophoretic patterns have been observed for the intermediate degradation products produced by other tissue proteinases, cathepsins D, B and G, though the size and hexosamine composition of the limit degradation product differed in each case. Thus it could be postulated that the initial cleavages in proteoglycan degradation are common to all proteinases, and that differences occur only in the final cleavages which produce the clusters of limiting size. I thank the Nuffield Foundation for financial support.
Hardingham, T. E., Ewins, R. J. F. & Muir, H. (1976) Bioclzem. J. 157, 127-143 Heinegard, D. & Hascall, V. C. (1974~)J . Biol. Chem. 249,4250-4256 Heinegard, D. & Hascall, V. C. (19746) Arch. Biochern. Biophys. 1 6 5 , 4 2 7 4 1 Keiser, H., Greenwald, R. A., Feinstein, G. & Janoff, A. (1976)J. Clin. Invest. 57,625-632 McDevitt, C . A. & Muir, H. (1971) Anal. Biochem. 44,612-622 Roughley, P. J. & Mason, R. M. (1976) Biochem. J. 157, 357-367 Sajdera, S. W. &. Hascall, V. C. (1969) J. Biol. Chem. 244, 77-87 Starkey, P. M. & Barrett, A. J. (1976) Biochem. J . 155, 255-263
Vol. 5
