Journul of Orthopuedic- Research 10:621630 Raven Press, Ltd., New York 0 1992 Orthopaedic Rebearch Society
Link Protein Shows Species Variation in Its Susceptibility to Proteolysis J. Liu, *J. D. Cassidy, *A. Allan, tP. J. Neame, J. S . Mort, and P. J. Roughley Shriners Hospital for Crippled Children and McGil1 University, Montreal, Quebec; *University o f Saskatchewan, Sashatoon, Canada; and PShriners Hospital for Crippled Children, Tampa, Florida, U.S.A.
Summary: Human cartilage link protein exists as three native components, while equine, bovine, and porcine cartilage link protein exist as two and Swarm rat chondrosarcoma link protein exists as only one component. These nonhuman link protein components represent intact protein structures, and there is little evidence for proteolytically modified forms in nonhuman tissues. In human cartilage, the proteolytic production of modified link proteins increases with age, whereas high amounts of such products were not seen in the nonhuman tissues. However, the small amounts of link protein fragments that were observed in the nonhuman cartilages were of a similar size to their human counterparts. On digestion of human proteoglycan aggregate with stromelysin. rapid modification of the link protein components occurred, whereas the aggregates from nonhuman cartilages showed incomplete cleavage of their link protein components. The relative resistance of nonhuman link protein to stromelysin may in part be due to a unique amino acid substitution present near the enzymic cleave site. Key Words: Link protein-Degradation-Age changes-Species variation-Stromelysin.
Proteoglycan subunits are essential components of hyaline cartilage, as they endow this tissue with its special property of compressive stiffness (19). Most of the proteoglycan subunits are able to form aggregates in the presence of other tissue components, namely, hyaluronate and link protein (15,16), and the resulting macromolecular complexes have been observed in situ (34) to be associated with collagen fibrils. It has been demonstrated that in the absence of link proteins the hyaluronateiproteoglycan subunit complex is not stable at low pH, elevated temperature, high ionic strength, or high centrifugal force (12,15,38). In addition, proteoglycan subunits in the link protein-free complex can be
dissociated in the presence of low molecular weight hyaluronate oligomers, but this is not possible in the link protein-stabilized aggregate (15,17). The presence of link proteins also serves to partially protect the hyaluronate binding region of the proteoglycan subunit from proteolytic attack, so enabling this region to be isolated (18). In previous studies, human link proteins have been shown to exist as three components, characterized as bands of M , 48,000,44,000, and 41,000 by SDS/polyacrylamide gel electrophoresis (SDS/ PAGE) in articular cartilage and intervertebral disc from individuals ranging in age from the fetus to the mature adult (26,32,36). These three components are termed LPl, LP2, and LP3, respectively. All human link protein components appear to be derived from the same protein core, but they differ in their degree of glycosylation and their protein core length. The LP1 differs from LP2 due to the pres-
Received August 28, 1991; accepted March 25, 1992. Address correspondence and reprint requests to Dr. J. Liu at the Joint Diseases Laboratory, Shriners Hospital for Crippled Children, 1529 Cedar Avenue, Montreal, Quebec I13G 1 A6, Canada.
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ence of an additional N-linked oligosaccharide substituted at residue 6. Both LP1 and LP2 have a protein core longer than that of LP3, which in the neonate results by the loss of a 16-amino acid residue peptide (22,30). This shortening of the core protein in LP3 is thought to be due to proteolytic cleavage of either of the two larger components near their N-termini (21). During aging, the structure and abundance of the human link protein components change, with LPl being more predominant in the young and LP3 increasing with agc (26). Moreover, in the adolescent and the adult, fragmentation of a portion of each link protein component occurs to yield products of M , 26,00&33,000 under reducing conditions. The degree of fragmentation shows a tendency to increase with increasing age (26). These age-related changes are thought to be the result of proteolytic processing (39). Interestingly, the metalloproteinase stromelysin specifically cleaves link protein between residues His-16 and Ile-17, a site identical to that accounting for the generation in situ of the smallest human link protein (LP3) from the two larger components (LP1 and LP2) in the neonate (30). In contrast, the production of the disulfide bond-stabilized fragmented link proteins has not been reproduced in vitro by physiologically relevant proteinases. However, products of a similar size have been obtained in vitro by the lysosomal cysteine proteinase, cathepsin L, and by the action of hydroxyl radicals (29,35). To date, link proteins have been described in a variety of cartilaginous tissues from different species. The link proteins from bovine nasal cartilage are generally reported as two components with molecular weights of about 44,000 and 48,000 (1,40), although a smaller componcnt has also been observed (2). The link proteins isolated from bovine articular cartilage appear to be identical to those from bovine nasal cartilage (42). Link proteins also exist as two major components in both chicken and rabbit cartilage (1 1,24). In contrast, link protein in the Swarm rat chondrosarcoma is only present as a single component, with a molecular 5ize comparable to the smaller of the two major link proteins present in bovine nasal cartilage (7,3 1). While most studies of maturation and age-related alterations in the structure of link protein have been carried out on human cartilage, there is evidence for similar changes also occurring in other species. For example, the proportion of smaller link protein components in rabbit articular cartilage has been
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reported to increase with age (11). However, it is not clear whether this definitely represents an increase in proteolytically modified forms equivalent to LP3. Little evidence for the presence of link protein frdgmentation in cartilage from other species has been reported, and it is possible that link protein from nonhuman species does not have a similar susceptibility to proteolytic degradation. Our study was undertaken to examine the link protein in proteoglycan aggregates from a variety of human and animal tissues to provide information about variation occurring in the abundance and structure of link protein degradation products between species and between sites in a given species. MATERIALS AND METHODS
Materials Human femoral head, femoral condylar, and humeral head articular cartilage, meniscus, and intervertebral discs (L4-L5 and L5-S l), which appeared macroscopically normal, were obtained at the time of autopsy and within 20 h of death. Equine femoral condylar articular cartilage was obtained from mixed breed horses at the time of autopsy and within 24 h of death. Bovine nasal septa were obtained from neonatal and mature animals. Pig cartilage proteoglycan aggregate was generously supplied by Dr. T. Hardingham (Kennedy Institute of Rheumatology, London, England). Swarm rat chondrosarcoma proteoglycan aggregate was generously supplied by Dr. Clive Roberts (Shriners Hospital, Montreal, Canada). Guanidinium chloride and trypsin (L-I-tosylamino-S-phenylethylchloromethylketone treated) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). CsCl was from Accurate Chemical and Scientific Co. (Westbury, NY, U.S.A.) Acrylamide, methylenebisacrylamide. SDS, and nitrocellulose membranes were from Bio-Rad Laboratories (Richmond, CA, U.S.A.). Immobilon membranes were from Millipore Corp. (Bedford, MA, U.S.A.). '251-labeled Protein A was from Amersham (Oakville, Ontario, Canada). Recombinant human interleukin-1 f3 was purchased from Cell Biology Boehringer (Boehringer Mannheim GmbH., Montreal, Canada). Hydrogen peroxide (SO%, vol/vol) was from Fisher Scientific (Montreal, Canada). Cathepsin L was from Aminotech (Nepean, Ontario, Canada). Monoclonal antibody 9/30/8-A-4 to link protein was
LINK PR 0TEIN DEGRADA TION
purchased from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, TA, U.S.A.). Preparation of Human Cartilage Prostromelysin Cartilage pieces (1-2 mm in each dimension) were cultured in serum-free medium (14) in the presence of 1 unit/ml of human recombinant interleukin-lg. The cultures were maintained for up to 14 days, with a change of fresh culture medium every 2 days. The latent neutral metalloproteinase activity in the culture medium was monitored by a L3H]caseindegradation assay following activation with p-aminophenylmercuric acetate (4,13). The metalloproteinase activity was concentrated by ultrafiltration using Amicon YM-5 membranes and separated from endogenous proteoglycan fragments b y gel filtration through Ultrogel AcA-45, as described by Campbell et al. (4), then reconcentrated. The purified prostromelysin was visualized as two distinct bands with molecular vizes of M , 56,000 and 58,000 by SDSIPAGE. Preparation of Tissue Proteins Proteins from human cartilage, meniscus, and intervertebral disc were prepared by extracting tissue pieces with 4 M guanidinium chloride and 0.1 M sodium acetate buffer, pH 6.0, containing proteinase inhibitors, at 4°C for 48 h. and subjecting the extract to dissociative CsCl density-gradient centrifugation using a starting density of 1.40 g/ml (38). The resulting density gradients were fractionated and monitored for absorbance at 280 nm, density, and uronic acid ( 3 ) . Protein was recovered in the fractions of lowest density (pooled as D3) and was dialyzed against 0.125 M TrisIHCl buffer, pH 6.8, then against the same buffer containing 0.1% SDS, before analysis by SDSIPAGE. Preparation of Proteoglycan Aggregates Human articular, equine articular, and bovine nasal cartilage proteoglycan aggregates and human intervertebral disc proteoglycan aggregates were prepared by extracting cartilage or disc pieces with 4 M guanidinium chloride and 0.1 M sodium acetate buffer, pH 6.0, containing proteinase inhibitors, at 4°C for 48 h, and subjecting the dialyzed extract to associative CsCl density-gradient centrifugation using a starting density of 1.60 gIml(37). Proteoglycan was recovered from the bottom of the gradient and
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was dialyzed exhaustively against 0.1 M sodium acetate buffer, pH 6.0, and H,O before freeze drying. In the case of the cow, link protein was prepared from a purified neonatal proteoglycan aggregate preparation as described by Tang et al. (40). Treatment of Proteoglycan Aggregates with Trypsin Proteoglycan aggregates (2 mgIml) were dissolved in 0.1 M TrisIHCl buffer, pH 7.4. Trypsin was added to a final concentration of enzyme, 5 pgimg of proteoglycan aggregate. The samples were incubated at 37°C for 4 h, then foybean trypsin inhibitor was added to a final concentration of inhibitor, 20 pgimg of aggregate, and the samples were dialyzed against 0.125 M TrisiHCl buffer, pH 6.8, containing 0.1% SDS. Treatment of Proteoglycan Aggregates with Stromelysin The purified stromelysin fractions from gel filtration were dialyzed into 0.1 M NaCl, 5 mM CaCI,, and 0.1 M TrisIHCl buffer, pH 7.0. Proteoglycan aggregates were dissolved in the same buffer at 4.0 mgIml. An equivalent volume of each solution (0.3 ml) was mixed together and incubated at 37°C in the presence of 1 mM p-aminophenylmercuric acetate. Sufficient stromelysin was present to give limit degradation of human proteoglycan aggregate within 24 h. At intervals of 2, 8, and 24 h, 0.2 ml of the digestion mixture was retrieved and the reaction was terminated by adding EDTA to a final concentration of 10 mM. Samples were dialyzed against 0.125 M TrisiHCl buffer, pH 6.8, containing 0.1% SDS. SDS/PAGE and Immunoblotting Digested proteoglycan samples were analyzed under reducing conditions by the method of King and Laemmli (20), using 10% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (41). The membranes were incubated in blocking solution [3% (wthol) bovine serum albumin in phosphate-buffered saline solution (10 mM sodiumipotassium phosphate buffer, pH 7.2, containing 0.145 M NaCl and 0.05% NaN,)] for 1 h at room temperature, then incubated for 4 h at room temperature in the blocking solution containing monoclonal antibody, 9/30/8-A-4. After washing with phosphate-buffered saline solution, the membrane was incubated for 1 h at room tem-
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perature in blocking solution containing "'I-labeled Protein A, then washed as described earlier. Link proteins were revealed by autoradiography using Kodak XAR film. Protein Sequence Analysis Individual equine link proteins were separated by SDSiPAGE, then electroblotted onto Immobilon membranes (23). Components identified by Coomassie Brilliant Blue staining were excised and analyzed directly using an Applied Biosystems model 470A sequencer equipped with an on-line model 120A analyzer to resolve phenylthiohydantoin derivatives of released amino acids, Purified total bovine link proteins were analyzed directly using an Applied Biosystems model 473A sequencer.
Comparison of Human, Equine, Bovine, Porcine, and Rat Link Protein
RESULTS Comparison of Human Cartilage, Meniscus, and Intervertebral Disc Link Protein To compare the variation in link protein heterogeneity at different sites, protein from various joints and intervertebral discs from the same adult were analyzed by SDSiPAGE followed by electroblotting onto nitrocellulose and subsequent visualization of link protein using the monoclonal antibody 9/30/8A-4 (Fig. I). Both the native link protein components and their fragmentation Droducts rnirrratpd
M~ lo3
46 -e
31 +
1
2
3
4
5
6
FIG. 1. Comparison of human cartilage, meniscus, and intervertebral disc link protein. An equal weight of protein (133) from the shoulder (lane l ) , hip (lane 2), knee (lane 3), meniscus (lane 4), nucleus pulposus (lane 5), and annulus fibrosus (lane 6) of the same individual was analyzed by S D S / polyacrylamide gel electrophoresis followed by immunoblotting. The positions of molecular weight markers are indicated (M, 46 x lo3, ovalbumin; M, 31 X lo3, carbonic anhydrase).
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with the same electrophoretic mobility for the different anatomical sites. However, the abundance of the link protein and the relative amount of the different components varied between the samples. Link protein was most abundant in the articular cartilages, with the greatest levels present in the hip. In the fibrocartilages. link protein was more abundant in the disc tissues than the meniscus. The ratio of LP1 to LP3 varied in the different tissues, with the relative amount of LP1 most prominent in the meniscus. In contrast, relative to the amount of LP1, the abundance of LP3 was greatest in the articular cartilages. Link protein fragmentation was present in all the tissues, though its relative abundance also varied. The proportion of fragmented link protein to total link protein present in the cartilage and disc tissues appeared higher than in the meniscus.
To compare the variation of link protein heterogeneity between species, proteoglycan aggregate from human, equine, bovine, and porcine cartilage and rat chondrosarcoma were analyzed by SDSJ PAGE (Fig. 2). While human cartilage proteoglycan aggregate showed three native link protein components, equine, bovine, and porcine proteoglycan aggregates exhibited only two components, with that of lower molecular size being less prominent in equine cartilage. The electrophoretic mobility of the larger link protein in equine cartilage was lower than that of LP1 in human cartilage, whereas the larger link protein in both bovine and porcine proteoglycan aggregates had a similar molecular size to LP1 in human cartilage. The smaller link protein in the nonhuman cartilages migrated to a position intermediate between that of the LP2 and LP3 components of human cartilage. In contrast, rat chondrosarcoma proteoglycan aggregate possessed only one link protein, migrating with an equivalent sire to LP2 in human proteoglycan aggregate. Unlike human link protein, no more mobile third component, equivalent to LP3, was observed in cartilage from any of the other species. Comparison of the N-Terminal Sequences of Human, Bovine, and Equine Native Link Protein To clarify the nature of the two native link protein components in the nonhuman cartilages, the N-terminal sequences of bovine and equine link proteins
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LINK PROTEIN DEGRADATION
1
10
Human : D H L S D N Y T L D . . . . .
-
LP1 + LP2 + LP3
Horse{
D H R S D - Y T L D , . . . . Upper band D H R S D N Y T L D . . . . . Lower band
FIG. 3. N-terminal sequences of bovine and equine link proteins. Bovine and equine link protein sequences are compared with the intact human link protein sequence. Bovine link protein was analyzed as a purified sample containing both link protein components. Equine link protein components were analyzed separately following electroblotting. The dash symbol (-) represents a blank cycle on sequencing, and the symbol n represents an unusually low yield of asparagine on sequencing.
from adult human, mature equine, and bovine cartilage were analyzed by SDSiPAGE (Fig. 4). Link protein fragments of similar size were evident from human (Fig. 4,lane l), equine (Fig. 4,lane 2), and bovine (Fig. 4, lane 3) cartilage, though fragmentation was much more abundant in human cartilage than in equine or bovine cartilage.
1
2
3
4
5
FIG. 2. Comparison of human, equine, bovine, porcine, and rat link protein. An equal weight of proteoglycan aggregate from the human (lane l ) , horse (lane 2), cow (lane 3), pig (lane 4), and rat (lane 5) was analyzed by SDS/polyacrylamide gel electrophoresis followed by immunoblotting. The positions of the three human link protein components (LPl, 2, and 3) are indicated.
were determined. After their separation by SDSi PAGE, it was found that the two equine cartilage link protein components were equivalent to human LP1 and LP2, with a blank cycle indicative of the presence of an additional N-linked oligosaccharide at residue 6 on the larger equine link protein. Analysis of a mixture of the two bovine link proteins was also indicative of components equivalent to human LP1 and LP2, with the asparagine at residue 6 appearing to be mostly glycosylated. There was no evidence for a proteolytically modified link protein equivalent to human LP3 in either the horse or cow (Fig. 3). The sequences were readable for at least 27 cycles in both cases.
Comparison of Link Protein Fragments from Human, Equine, and Bovine Cartilage To compare the variation of link protein fragments between species, proteoglycan aggregates
A
Mr x I $
-
46 -F
31
1
2
3
FIG. 4. Comparison of link protein fragments from human, equine, and bovine cartilage. An equal weight of proteoglycan aggregate from the human (lane l), horse (lane 2), and cow (lane 3) was analyzed by SDSipolyacrylamide gel electrophoresis followed by immunoblotting. The equine and bovine samples were overexposed to visualize the fragmentation of link protein. The positions of molecular weight markers are indicated (M, 46 x lo3, ovalbumin: M, 31 x lo3, carbonic anhydrase).
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Comparison of Age-Related Changes in Equine Link Protein Previous study of human link protein demonstrated the accumulation of the smallest link protein component, LP3, and internal link protein fragmentation with increasing age. To determine whether similar age-related changes may occur in nonhuman species, a serial age group of equine cartilage was analyzed by SDSIPAGE (Fig. 5). In young horses, two intact link protein components were observed. After the animals reached their second decade, a third more mobile intact link protein was apparent,
] Intact LP - LP fragments
1234 5 6 7 8 FIG. 6. Comparison of age-related changes in equine link protein fragments. Protein from an equal weight of tissue from different horses was analyzed by SDSlpolyacrylarnide gel electrophoresis followed by irnmunoblotting. Samples are from horses aged 2 days (lane l), 12 days (land 2), 8 months (lane 3),1 year (land 4), 4 years (lane 5), 8 years (lane 6),15-20 years (lane 7),and 28 years (lane 8).The positions of the intact LP and LP fragments are indicated. Blots are overexposed to enhance visualization of LP fragments.
46-
possibly equivalent to an LP3-like molecule in the human. However, this LP3-like molecule in equine cartilage was never a major form, unlike LP3 in adult human cartilage. In addition, unlike the situation in human link protein, the ratio of the largest to the smaller link proteins did not change markedly with age. When immunoblots were overexposed, link protein fragments were observed, and their abundance was found to increase with the age of the animals (Fig. 6). However, w e n in the mature adult the fragments represented only a minor proportion of the total molecule.
31+
1
2
3
4
5
FIG. 5. Comparison of age-related changes in equine link protein. An equal weight of proteoglycan aggregate from animals aged 2 days (lane l),1 year (lane 2),4 years (lane 3), 15-20 years (lane 4),and 28 years (land 5) was analyzed by SDS/polyacrylamide gel electrophoresis followed by immunoblotting. The positions of molecular weight markers are indicated (Mr 46 x lo3,ovalbumin; M , 31 x lo3,carbonic an hydrase).
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Comparison of the Effects of Trypsin Treatment on Link Protein in Human, Equine, Bovine, Pig, and Rat Proteoglycan Aggregates Because little link protein similar to LP3 was detected in the mature equine and bovine cartilage, and because the human LP3 is known to be a product of proteolysis, one might presume that either
LINK PROTEIN DEGRADATION there is less proteinase activity available to act on the link protein in the nonhuman species, or that link protein from the nonhuman species is relatively resistant to proteolytic modification. As LP3 generation occurs in the N-terminal region of the link protein between the oligosaccharide attachment sites, it is possible that variation in oligosaccharide structure and size could sterically interfere with the access of proteinases. To test this possibility, human, horse, bovine, pig, and rat proteoglycan aggregates were incubated with trypsin. In all cases, link protein was completely converted to a single LP3-like form (Fig. 7), suggesting that there is no major impediment to proteinase access in the N-terminal region of the link protein molecules. The absence of a native link protein of similar mobility to the trypsin-generated link protein in the nonhuman species provides further evidence for the absence of LP3-like molecules in these species.
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determine whether this enzyme can cleave link protein from other species, the action of human cartilage stromelysin on the link protein in various proteoglycan aggregates was investigated (Fig. 8). After 24 h of incubation, human link protein was completely degraded by stromelysin to yield a single protein component that comigrates with the native LP3 component. However, the aggregates from nonhuman species, particularly in the case of the horse, showed incomplete cleavage of their link protein components. Rat link protein seemed more prone to stromelysin degradation than that of the other nonhuman species. The degraded link proteins produced by the action of stromelysin in rat, bovine, and porcine proteoglycan aggregates had mobilities comparable to that of human LP3. DISCUSSION In our study, it was shown that under electrophoretic conditions where human link protein exhibits three components, equine, bovine, and pig link protein exist as two components, and rat chondrosarcoma link protein exists as a single component. Two link protein components have also been reported in chicken xyphoid cartilage (24), canine articular cartilage (lo), and rabbit articular cartilage
Comparison of the Effects of Stromelysin Treatment on Link Protein in Human, Equine, Bovine, Pig, and Rat Proteoglycan Aggregates Previous evidence suggests that human LP3 (particularly in the neonate) is predominately a degradation product due to the action of stromelysin. To
FIG. 7. Susceptibility of link protein to trypsin digestion. Proteoglycan aggregates from the human, horse, cow, pig, and rat were analyzed directly ( - ) or following trypsin treatment ( + ) by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting.
Trypsin
,-
+,
Human
,-
+I
Horse
,-
+I
Bovine
,-
+ , I - +, Pig
Rat
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FIG. 8. Susceptibility of link protein to stromelysin digestion. Proteoglycan aggregates from human, horse, cow, pig, and rat were analyzed directly ( - ) or following treatment with stromelysin ( + ) by SDSipolyacrylamide gel electrophoresis and subsequent immunoblotting.
(33). In human cartilage, the link proteins have been defined as LPI, LP2, and LP3, based on their increasing electrophoretic mobility on SDSiPAGE. This electrophoretic heterogeneity is due to structural variation in oligosaccharide substitution (for LPl and LP2) and proteolytic modification (for LP3) (25). When two link protein components are observed in other species they may represent LP1 and LP2, LP2 and LP3, or even LPI and LP3, in comparison with the three components of human link protein. When a single link protein component is observed, it could theoretically be equivalent to any of the three forms of the human link protein. Thus, it would be desirable to have a standard nomenclature whereby link protein components can be defined on the basis of common structural features. An intact core protein with two N-linked oligosaccharides would be an LPl form, an intact core protein with one N-linked oligosaccharide would be an LP2 form, and proteolytic modification between the sites of oligosaccharide substitution would yield an LP3 form (30). On this system, the two equine link protein components and those in bovine (2) and pig cartilage (8) are LPl and LP2 forms, the single component of link protein in rat chondrosarcoma is an LP2 form, and LP3 forms are not commonly present in nonhuman species. The present data based on the electrophoretic mobility of native and trypsin-treated link proteins would indicate that while the N-terminal oligosaccharide substitution site is not always occupied, that at residue 41 is never free. The abundance of human cartilage LP3 and link
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protein fragments increases with the age of the individual (26). This is thought to be due to continual proteolytic modification of the link proteins within the extracellular matrix throughout life and the accumulation of these modified forms with age because of their continued interaction with other cartilage matrix components. In human cartilage, the enzymatic production of LP3 and link protein fragments seems to be related, since an increase in the abundance of LP3 is generally accompanied by an increase in the abundance of link protein fragments, though it is likely that different agents are involved. In this study the two extremes are represented by the hip and the meniscus, which show high and low link protein modification, respectively. In contrast, high amounts of LP3 and link protein fragments were never seen in the nonhuman cartilages, irrespective of age. Although an increase in the abundance of link protein fragments was observed with age in horses, the amount of fragmented link protein was considerably less than that found with human link protein. Thus, while the mechanisms controlling proteolytic modification of link protein might be similar in the different species, they occur to a much lower degree in the nonhuman species. One explanation for the low abundance of modified link proteins in the horse and cow, even in the mature age group, could be the relatively long life span of the human in comparison to the other species. Such longevity would favor the accumulation of structurally stable link protein degradation products. Indeed, there is no evidence to indicate that the proteolytically modified forms of link protein are less
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LINK PR 0TElN DEGRA D A T I 0N functional than their intact counterparts in their abi1i ty to form proteogl y can aggregates . Alternative explanations to account for the low proteolytic modification of nonhuman link protein are also possible. As proteolysis to generate LP3 forms occurs in the vicinity of the oligosaccharides, variation in oligosaccharide structure between the species might sterically hinder the access of proteinases. However, proteolytically modified link proteins equivalent to LP3 have been generated in vitro by trypsin (9) or clostripain ( 5 )digestion of the LPI and LP2 forms in the nonhuman species. This, together with our data on trypsin degradation of link protein from various species, indicates that there is no major impediment to proteinase access in the different link proteins. In contrast, species differences in susceptibility were observed with stromelysin. Whether this is related to the relatively large size of stromelysin is not known, but it is also possible that the difference could be related to the primary amino acid structure of the link protein. In general, the link protein sequence is highly conserved between species with the exception of the extreme N-terminus of the molecule, which is removed in the proteolytic generation of the LP3 forms. In the region where stromelysin cleaves, there is a unique amino acid substitution in the human sequence (Fig. 9) (27), which falls within the six amino acid sequence spanning the cleavage site thought to be recognized by proteinases (39). This could contribute to the enhanced susceptibility of human link protein to stromelysin cleavage. However, one cannot discount other alternative explanations at this time, as immunological evidence suggests that the folding of the protein in this region is complex (6). It is also possible that stromelysin from different species could show variation in substrate specificity, or that stromelysin is secreted or activated to a much greater degree in human cartilage than in other species. Regardless of the origin of the differences, the use of link protein as a con-
Human
t
1 10 2o D H L S D ~ T L D H D R ~ I H I O A E N G P H L L V
Bovine Horse
D H H S D N Y T V D H D R V I H I Q A E N G P R L L V
Pig
D H L S N N Y T L D H D R V I H I Q A E N G P R L L V
Rat
D H L S D S Y T P D Q D R V I H I Q A E N G P R L L V
D H R s D TE
L D H D
R
v
I
H I
Q A E N
G P R L L
v
FIG. 9. N-terminal sequences of link protein from different species. Data for human (8), horse, pig (€9,cow, and rat (27) are depicted. The symbols used are underline, a potential site for N-linked oligosaccharide substitution; square, emphasis of the residue change in the human: and solid arrow, site of stromelysin cleavage.
venient indicator of proteolysis in cartilage during aging (28) does not appear to be applicable to other species. Acknowledgment: We thank the departments of Pathology at t h e Royal Victoria Hospital a n d the Montreal General Hospital for the provision of autopsy facilities, Ms. Elisa de Miguel for operation of the protein sequencer, and Ms. J a n e Wishart for preparing the figures. This work was supported b y the Arthritis Society of Canada and the
Shriners of North America.
REFERENCES 1. Baker JR, Caterson B: The isolation of “link protein” from bovine nasal cartilage. Biochim Biophys Acta 532:24%258, 1978.
2. Baker JR, Caterson B: The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage. J Rial Chem 254:2387-2393, 1979. 3. Bitter T, Muir HM: A modified uronic acid carbazole reaction. Anal Biochern 4:330-334, 1962. 4. Campbell IK. Golds EE, Mort JS, Roughley PJ: Human articular cartilage secretes characteristic metal dependent proteinases upon stimulation by mononuclear cell factor. J Rheumntol 13:20-27, 1986. 5 . Caputo CB, MacCallum DK, Kimura JH, Hascall VC: Characterization of fragments produced by clostripan digestion of proteoglycans from the Swarm rat chondrosarcoma. Arch Biochem Biuphys 204:220-233, 1980. 6. Caterson B , Baker JR, Christner JE, Lee Y, Lentz M: Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan. J Biol Chem 260:11348-11356, 1985. 7. Caterson B, Baker JR, Levitt D, Paslay JW: Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan. J B i d Chem 254:9369-9372, 1979. 8. Dudhia J , Hardingham TE: Isolation and sequence of cDNA clones for pig and human cartilage link protein. J Mol Biol 2061749-754, 1989. 9. Faltz LL, Caputo CB. Kimura JH. Schrode J, Hascall VC: Structure of the complex between hyaluronic acid, the hyaluronic acid-binding region, and the link protein of proteoglycan aggregates from the Swarm rat chondrosarcoma. J Biol Chem 254:1381-1387, 1979. 10. Fife RS, Caterson B . Myers SL: Identification of link proteins in canine synovial cell cultures and canine articular cartilage. J Cell B i d 100:1050-1055, 1985. 11. Flannery CR, Urbanek PJ, Sandy JD: The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage. J Orthop Res 8:78-85, 1990. 12. Franzen A, Bjornsson S, Heinegird D: Cartilage proteoglycan aggregate formation. Biochem J 197:669474, 1981. 13. Golds EE, Santer V, Killackey J. Koughley PJ: Mononuclear cell factors stimulate the concomitant secretion of distinct latent proteoglycan, gelatin and collagen degrading enzymes from human skin fibroblasts and synovial cells. J Rheumntol 10:861-871, 1983. 14. Handley CJ, Lowther DA: Extracellular matrix metabolism by chondrocytes. 111. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in culture. Biochim Biophys Acta 500:132-13Y, 1977. 15. Hardingham TE: The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J 177:237-247, 1979.
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16. Hascall VC: Interaction of cartilage proteoglycans with hyaluronic acid. J Supramol Struct 7:lOl-120, 1977. 17. Hascall VC, Heinegird D: Aggregation of cartilage proteoglycan. TI. Oligosaccharide competitors of the proteoglycanhyaluronic acid interaction. J Biol Clzein 249:42424249, 1974. 18. Heinegkrd D,Hascall VC: Aggregation of cartilage proteoglycans. 111. Characteristics of the proteins isolated from trypsin digests of aggregates. J Biol Chern 249:42504256, 1974. 19. Kempson GE, Muir H , Freeman MAR. Swanson SAV: Correlations between stiffness and the chemical constituents of cartilage on the human femoral head. Biochim Biophys Acta 215:?0-77,1970. 20. King J, Laemmli UK: Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 62:465-477, 1971. 21. Le Gltdic S, Ptrin JP, Bonnet F, Jolles P: ldentity of the protein cores of the two link proteins from bovine nasal cartilage proteoglycan complex. J Biol Chein 258:1475914761, 1983. 22. Liu J, Roughley PJ, Mort JS: Identification of human intervertebral disc stromelysin and its involvement in matrix degradation. J Orthop Res 9568-575, 1991. 23. Matsudaira P: Sequence from picomolc quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chern 262:1OO3S-10038. 1987. 24. McKeown-Longo PJ, Sparks KJ, Goetinck PF: Preparation and characterization of an antiserum against purified proteoglycan link proteins from avian cartilage. Coll Rda t Res 2~231-244,1982. 25. Mort JS. Caterson B, Poole AR, Roughley PJ: The origin of human cartilage proteoglycan link protein heterogeneity and fragmentation during aging. Biochem J 232:805-812,1985. 26. Mort JS, Poole AR, Roughley PJ: Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage. Riochem J 214:269-272. 1983. 27. Neame PJ, Christner JE, Bakcr JR: The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate. J Biol Chem 261:3519-3535, 1986. 28. h'guyen Q , Liu J, Roughley PJ, Mort JS: Link protein as an in situ monitor of endogenous proteolysis in adult human articular cartilage. Biochem J 278:143-147. 1991. 29. Nguyen Q,Mort JS. Roughley PJ: Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B. Biochem J 266:56%573, 1990.
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30. Nguyen Q,Murphy G, Roughley PJ. Mort JS: Degl-adation of proteoglycan aggregate by a cartilage melalloproteinase. evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ. Biochem J 259:6167, 1989. 31. Oegema TK Jr. Hascall VC, Dziewiatkowski DD: Isolation and characterization of proteoglycans from the Swarm rat chondrosarcoma. J Biol Chern 250:6151-6159, 1975. 32. Pearce RH, Mathieson JM, Mort JS, Roughley PJ: Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc. J Orrhop Res 7:861-867, 1989. 33. Plaas AHK, Sandy JD, Kimura JH: Biosynthehis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits. J Biol Chem 263:756& 7566, 1988. 34. Poole AR, Pidoux I, Reiner A, Rosenberg L: An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J Cell B i d 93:91C-920, 1982. 35. Roberts CK, Roughley PJ, Mort JS: Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Biochem J 259:805-411. 1989. 36. Roughley PJ, Morl JS: Aging and the aggregating proteoglycans of human articular cartilage. Clin Sci 71:337-344, 1986. 37. Roughley PJ. Poole AR, Mort JS: The heterogeneity of link proteins isolated from human articular cartilage proteoglycan aggregates. J Riol Chew 257:11908-11914,1982. 38. Roughley PJ, While RJ: Dermatan sulphate proteoglycans of human articular cartilage. The properties of dcrmatan sulphate proteoglycans I and 11. Biochetn J 262:823-827, 1989. 39. Schechter I, Berger A: On the size of the active site in proteases. 1. Papain. Biochem Biophys Res Conimrrn 27:157162, 1967. 40. Tang LH. Kosenbcrg L, Keiner A, Poole AR: Proteoglycans from bovine nasal cartilage. Properties of a soluble form of link protein. J Biol Chenz 254:10523-10531, 1979. 41. Towbin H , Staehelin T. Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Pror h'rrtl Acud Sci USA 76:435&4354, 1979. 42. Treadwell RV, Mankin DP. Ho PK, Mankin HJ: Cell-free synthesis of cartilage proteins: partial identification of proteoglycan core and link proteins. Biochemistry 19:22692275. 1980.
Link Protein Shows Species Variation in Its Susceptibility to Proteolysis J. Liu, *J. D. Cassidy, *A. Allan, tP. J. Neame, J. S . Mort, and P. J. Roughley Shriners Hospital for Crippled Children and McGil1 University, Montreal, Quebec; *University o f Saskatchewan, Sashatoon, Canada; and PShriners Hospital for Crippled Children, Tampa, Florida, U.S.A.
Summary: Human cartilage link protein exists as three native components, while equine, bovine, and porcine cartilage link protein exist as two and Swarm rat chondrosarcoma link protein exists as only one component. These nonhuman link protein components represent intact protein structures, and there is little evidence for proteolytically modified forms in nonhuman tissues. In human cartilage, the proteolytic production of modified link proteins increases with age, whereas high amounts of such products were not seen in the nonhuman tissues. However, the small amounts of link protein fragments that were observed in the nonhuman cartilages were of a similar size to their human counterparts. On digestion of human proteoglycan aggregate with stromelysin. rapid modification of the link protein components occurred, whereas the aggregates from nonhuman cartilages showed incomplete cleavage of their link protein components. The relative resistance of nonhuman link protein to stromelysin may in part be due to a unique amino acid substitution present near the enzymic cleave site. Key Words: Link protein-Degradation-Age changes-Species variation-Stromelysin.
Proteoglycan subunits are essential components of hyaline cartilage, as they endow this tissue with its special property of compressive stiffness (19). Most of the proteoglycan subunits are able to form aggregates in the presence of other tissue components, namely, hyaluronate and link protein (15,16), and the resulting macromolecular complexes have been observed in situ (34) to be associated with collagen fibrils. It has been demonstrated that in the absence of link proteins the hyaluronateiproteoglycan subunit complex is not stable at low pH, elevated temperature, high ionic strength, or high centrifugal force (12,15,38). In addition, proteoglycan subunits in the link protein-free complex can be
dissociated in the presence of low molecular weight hyaluronate oligomers, but this is not possible in the link protein-stabilized aggregate (15,17). The presence of link proteins also serves to partially protect the hyaluronate binding region of the proteoglycan subunit from proteolytic attack, so enabling this region to be isolated (18). In previous studies, human link proteins have been shown to exist as three components, characterized as bands of M , 48,000,44,000, and 41,000 by SDS/polyacrylamide gel electrophoresis (SDS/ PAGE) in articular cartilage and intervertebral disc from individuals ranging in age from the fetus to the mature adult (26,32,36). These three components are termed LPl, LP2, and LP3, respectively. All human link protein components appear to be derived from the same protein core, but they differ in their degree of glycosylation and their protein core length. The LP1 differs from LP2 due to the pres-
Received August 28, 1991; accepted March 25, 1992. Address correspondence and reprint requests to Dr. J. Liu at the Joint Diseases Laboratory, Shriners Hospital for Crippled Children, 1529 Cedar Avenue, Montreal, Quebec I13G 1 A6, Canada.
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ence of an additional N-linked oligosaccharide substituted at residue 6. Both LP1 and LP2 have a protein core longer than that of LP3, which in the neonate results by the loss of a 16-amino acid residue peptide (22,30). This shortening of the core protein in LP3 is thought to be due to proteolytic cleavage of either of the two larger components near their N-termini (21). During aging, the structure and abundance of the human link protein components change, with LPl being more predominant in the young and LP3 increasing with agc (26). Moreover, in the adolescent and the adult, fragmentation of a portion of each link protein component occurs to yield products of M , 26,00&33,000 under reducing conditions. The degree of fragmentation shows a tendency to increase with increasing age (26). These age-related changes are thought to be the result of proteolytic processing (39). Interestingly, the metalloproteinase stromelysin specifically cleaves link protein between residues His-16 and Ile-17, a site identical to that accounting for the generation in situ of the smallest human link protein (LP3) from the two larger components (LP1 and LP2) in the neonate (30). In contrast, the production of the disulfide bond-stabilized fragmented link proteins has not been reproduced in vitro by physiologically relevant proteinases. However, products of a similar size have been obtained in vitro by the lysosomal cysteine proteinase, cathepsin L, and by the action of hydroxyl radicals (29,35). To date, link proteins have been described in a variety of cartilaginous tissues from different species. The link proteins from bovine nasal cartilage are generally reported as two components with molecular weights of about 44,000 and 48,000 (1,40), although a smaller componcnt has also been observed (2). The link proteins isolated from bovine articular cartilage appear to be identical to those from bovine nasal cartilage (42). Link proteins also exist as two major components in both chicken and rabbit cartilage (1 1,24). In contrast, link protein in the Swarm rat chondrosarcoma is only present as a single component, with a molecular 5ize comparable to the smaller of the two major link proteins present in bovine nasal cartilage (7,3 1). While most studies of maturation and age-related alterations in the structure of link protein have been carried out on human cartilage, there is evidence for similar changes also occurring in other species. For example, the proportion of smaller link protein components in rabbit articular cartilage has been
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reported to increase with age (11). However, it is not clear whether this definitely represents an increase in proteolytically modified forms equivalent to LP3. Little evidence for the presence of link protein frdgmentation in cartilage from other species has been reported, and it is possible that link protein from nonhuman species does not have a similar susceptibility to proteolytic degradation. Our study was undertaken to examine the link protein in proteoglycan aggregates from a variety of human and animal tissues to provide information about variation occurring in the abundance and structure of link protein degradation products between species and between sites in a given species. MATERIALS AND METHODS
Materials Human femoral head, femoral condylar, and humeral head articular cartilage, meniscus, and intervertebral discs (L4-L5 and L5-S l), which appeared macroscopically normal, were obtained at the time of autopsy and within 20 h of death. Equine femoral condylar articular cartilage was obtained from mixed breed horses at the time of autopsy and within 24 h of death. Bovine nasal septa were obtained from neonatal and mature animals. Pig cartilage proteoglycan aggregate was generously supplied by Dr. T. Hardingham (Kennedy Institute of Rheumatology, London, England). Swarm rat chondrosarcoma proteoglycan aggregate was generously supplied by Dr. Clive Roberts (Shriners Hospital, Montreal, Canada). Guanidinium chloride and trypsin (L-I-tosylamino-S-phenylethylchloromethylketone treated) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). CsCl was from Accurate Chemical and Scientific Co. (Westbury, NY, U.S.A.) Acrylamide, methylenebisacrylamide. SDS, and nitrocellulose membranes were from Bio-Rad Laboratories (Richmond, CA, U.S.A.). Immobilon membranes were from Millipore Corp. (Bedford, MA, U.S.A.). '251-labeled Protein A was from Amersham (Oakville, Ontario, Canada). Recombinant human interleukin-1 f3 was purchased from Cell Biology Boehringer (Boehringer Mannheim GmbH., Montreal, Canada). Hydrogen peroxide (SO%, vol/vol) was from Fisher Scientific (Montreal, Canada). Cathepsin L was from Aminotech (Nepean, Ontario, Canada). Monoclonal antibody 9/30/8-A-4 to link protein was
LINK PR 0TEIN DEGRADA TION
purchased from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, TA, U.S.A.). Preparation of Human Cartilage Prostromelysin Cartilage pieces (1-2 mm in each dimension) were cultured in serum-free medium (14) in the presence of 1 unit/ml of human recombinant interleukin-lg. The cultures were maintained for up to 14 days, with a change of fresh culture medium every 2 days. The latent neutral metalloproteinase activity in the culture medium was monitored by a L3H]caseindegradation assay following activation with p-aminophenylmercuric acetate (4,13). The metalloproteinase activity was concentrated by ultrafiltration using Amicon YM-5 membranes and separated from endogenous proteoglycan fragments b y gel filtration through Ultrogel AcA-45, as described by Campbell et al. (4), then reconcentrated. The purified prostromelysin was visualized as two distinct bands with molecular vizes of M , 56,000 and 58,000 by SDSIPAGE. Preparation of Tissue Proteins Proteins from human cartilage, meniscus, and intervertebral disc were prepared by extracting tissue pieces with 4 M guanidinium chloride and 0.1 M sodium acetate buffer, pH 6.0, containing proteinase inhibitors, at 4°C for 48 h. and subjecting the extract to dissociative CsCl density-gradient centrifugation using a starting density of 1.40 g/ml (38). The resulting density gradients were fractionated and monitored for absorbance at 280 nm, density, and uronic acid ( 3 ) . Protein was recovered in the fractions of lowest density (pooled as D3) and was dialyzed against 0.125 M TrisIHCl buffer, pH 6.8, then against the same buffer containing 0.1% SDS, before analysis by SDSIPAGE. Preparation of Proteoglycan Aggregates Human articular, equine articular, and bovine nasal cartilage proteoglycan aggregates and human intervertebral disc proteoglycan aggregates were prepared by extracting cartilage or disc pieces with 4 M guanidinium chloride and 0.1 M sodium acetate buffer, pH 6.0, containing proteinase inhibitors, at 4°C for 48 h, and subjecting the dialyzed extract to associative CsCl density-gradient centrifugation using a starting density of 1.60 gIml(37). Proteoglycan was recovered from the bottom of the gradient and
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was dialyzed exhaustively against 0.1 M sodium acetate buffer, pH 6.0, and H,O before freeze drying. In the case of the cow, link protein was prepared from a purified neonatal proteoglycan aggregate preparation as described by Tang et al. (40). Treatment of Proteoglycan Aggregates with Trypsin Proteoglycan aggregates (2 mgIml) were dissolved in 0.1 M TrisIHCl buffer, pH 7.4. Trypsin was added to a final concentration of enzyme, 5 pgimg of proteoglycan aggregate. The samples were incubated at 37°C for 4 h, then foybean trypsin inhibitor was added to a final concentration of inhibitor, 20 pgimg of aggregate, and the samples were dialyzed against 0.125 M TrisiHCl buffer, pH 6.8, containing 0.1% SDS. Treatment of Proteoglycan Aggregates with Stromelysin The purified stromelysin fractions from gel filtration were dialyzed into 0.1 M NaCl, 5 mM CaCI,, and 0.1 M TrisIHCl buffer, pH 7.0. Proteoglycan aggregates were dissolved in the same buffer at 4.0 mgIml. An equivalent volume of each solution (0.3 ml) was mixed together and incubated at 37°C in the presence of 1 mM p-aminophenylmercuric acetate. Sufficient stromelysin was present to give limit degradation of human proteoglycan aggregate within 24 h. At intervals of 2, 8, and 24 h, 0.2 ml of the digestion mixture was retrieved and the reaction was terminated by adding EDTA to a final concentration of 10 mM. Samples were dialyzed against 0.125 M TrisiHCl buffer, pH 6.8, containing 0.1% SDS. SDS/PAGE and Immunoblotting Digested proteoglycan samples were analyzed under reducing conditions by the method of King and Laemmli (20), using 10% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (41). The membranes were incubated in blocking solution [3% (wthol) bovine serum albumin in phosphate-buffered saline solution (10 mM sodiumipotassium phosphate buffer, pH 7.2, containing 0.145 M NaCl and 0.05% NaN,)] for 1 h at room temperature, then incubated for 4 h at room temperature in the blocking solution containing monoclonal antibody, 9/30/8-A-4. After washing with phosphate-buffered saline solution, the membrane was incubated for 1 h at room tem-
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perature in blocking solution containing "'I-labeled Protein A, then washed as described earlier. Link proteins were revealed by autoradiography using Kodak XAR film. Protein Sequence Analysis Individual equine link proteins were separated by SDSiPAGE, then electroblotted onto Immobilon membranes (23). Components identified by Coomassie Brilliant Blue staining were excised and analyzed directly using an Applied Biosystems model 470A sequencer equipped with an on-line model 120A analyzer to resolve phenylthiohydantoin derivatives of released amino acids, Purified total bovine link proteins were analyzed directly using an Applied Biosystems model 473A sequencer.
Comparison of Human, Equine, Bovine, Porcine, and Rat Link Protein
RESULTS Comparison of Human Cartilage, Meniscus, and Intervertebral Disc Link Protein To compare the variation in link protein heterogeneity at different sites, protein from various joints and intervertebral discs from the same adult were analyzed by SDSiPAGE followed by electroblotting onto nitrocellulose and subsequent visualization of link protein using the monoclonal antibody 9/30/8A-4 (Fig. I). Both the native link protein components and their fragmentation Droducts rnirrratpd
M~ lo3
46 -e
31 +
1
2
3
4
5
6
FIG. 1. Comparison of human cartilage, meniscus, and intervertebral disc link protein. An equal weight of protein (133) from the shoulder (lane l ) , hip (lane 2), knee (lane 3), meniscus (lane 4), nucleus pulposus (lane 5), and annulus fibrosus (lane 6) of the same individual was analyzed by S D S / polyacrylamide gel electrophoresis followed by immunoblotting. The positions of molecular weight markers are indicated (M, 46 x lo3, ovalbumin; M, 31 X lo3, carbonic anhydrase).
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with the same electrophoretic mobility for the different anatomical sites. However, the abundance of the link protein and the relative amount of the different components varied between the samples. Link protein was most abundant in the articular cartilages, with the greatest levels present in the hip. In the fibrocartilages. link protein was more abundant in the disc tissues than the meniscus. The ratio of LP1 to LP3 varied in the different tissues, with the relative amount of LP1 most prominent in the meniscus. In contrast, relative to the amount of LP1, the abundance of LP3 was greatest in the articular cartilages. Link protein fragmentation was present in all the tissues, though its relative abundance also varied. The proportion of fragmented link protein to total link protein present in the cartilage and disc tissues appeared higher than in the meniscus.
To compare the variation of link protein heterogeneity between species, proteoglycan aggregate from human, equine, bovine, and porcine cartilage and rat chondrosarcoma were analyzed by SDSJ PAGE (Fig. 2). While human cartilage proteoglycan aggregate showed three native link protein components, equine, bovine, and porcine proteoglycan aggregates exhibited only two components, with that of lower molecular size being less prominent in equine cartilage. The electrophoretic mobility of the larger link protein in equine cartilage was lower than that of LP1 in human cartilage, whereas the larger link protein in both bovine and porcine proteoglycan aggregates had a similar molecular size to LP1 in human cartilage. The smaller link protein in the nonhuman cartilages migrated to a position intermediate between that of the LP2 and LP3 components of human cartilage. In contrast, rat chondrosarcoma proteoglycan aggregate possessed only one link protein, migrating with an equivalent sire to LP2 in human proteoglycan aggregate. Unlike human link protein, no more mobile third component, equivalent to LP3, was observed in cartilage from any of the other species. Comparison of the N-Terminal Sequences of Human, Bovine, and Equine Native Link Protein To clarify the nature of the two native link protein components in the nonhuman cartilages, the N-terminal sequences of bovine and equine link proteins
625
LINK PROTEIN DEGRADATION
1
10
Human : D H L S D N Y T L D . . . . .
-
LP1 + LP2 + LP3
Horse{
D H R S D - Y T L D , . . . . Upper band D H R S D N Y T L D . . . . . Lower band
FIG. 3. N-terminal sequences of bovine and equine link proteins. Bovine and equine link protein sequences are compared with the intact human link protein sequence. Bovine link protein was analyzed as a purified sample containing both link protein components. Equine link protein components were analyzed separately following electroblotting. The dash symbol (-) represents a blank cycle on sequencing, and the symbol n represents an unusually low yield of asparagine on sequencing.
from adult human, mature equine, and bovine cartilage were analyzed by SDSiPAGE (Fig. 4). Link protein fragments of similar size were evident from human (Fig. 4,lane l), equine (Fig. 4,lane 2), and bovine (Fig. 4, lane 3) cartilage, though fragmentation was much more abundant in human cartilage than in equine or bovine cartilage.
1
2
3
4
5
FIG. 2. Comparison of human, equine, bovine, porcine, and rat link protein. An equal weight of proteoglycan aggregate from the human (lane l ) , horse (lane 2), cow (lane 3), pig (lane 4), and rat (lane 5) was analyzed by SDS/polyacrylamide gel electrophoresis followed by immunoblotting. The positions of the three human link protein components (LPl, 2, and 3) are indicated.
were determined. After their separation by SDSi PAGE, it was found that the two equine cartilage link protein components were equivalent to human LP1 and LP2, with a blank cycle indicative of the presence of an additional N-linked oligosaccharide at residue 6 on the larger equine link protein. Analysis of a mixture of the two bovine link proteins was also indicative of components equivalent to human LP1 and LP2, with the asparagine at residue 6 appearing to be mostly glycosylated. There was no evidence for a proteolytically modified link protein equivalent to human LP3 in either the horse or cow (Fig. 3). The sequences were readable for at least 27 cycles in both cases.
Comparison of Link Protein Fragments from Human, Equine, and Bovine Cartilage To compare the variation of link protein fragments between species, proteoglycan aggregates
A
Mr x I $
-
46 -F
31
1
2
3
FIG. 4. Comparison of link protein fragments from human, equine, and bovine cartilage. An equal weight of proteoglycan aggregate from the human (lane l), horse (lane 2), and cow (lane 3) was analyzed by SDSipolyacrylamide gel electrophoresis followed by immunoblotting. The equine and bovine samples were overexposed to visualize the fragmentation of link protein. The positions of molecular weight markers are indicated (M, 46 x lo3, ovalbumin: M, 31 x lo3, carbonic anhydrase).
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Comparison of Age-Related Changes in Equine Link Protein Previous study of human link protein demonstrated the accumulation of the smallest link protein component, LP3, and internal link protein fragmentation with increasing age. To determine whether similar age-related changes may occur in nonhuman species, a serial age group of equine cartilage was analyzed by SDSIPAGE (Fig. 5). In young horses, two intact link protein components were observed. After the animals reached their second decade, a third more mobile intact link protein was apparent,
] Intact LP - LP fragments
1234 5 6 7 8 FIG. 6. Comparison of age-related changes in equine link protein fragments. Protein from an equal weight of tissue from different horses was analyzed by SDSlpolyacrylarnide gel electrophoresis followed by irnmunoblotting. Samples are from horses aged 2 days (lane l), 12 days (land 2), 8 months (lane 3),1 year (land 4), 4 years (lane 5), 8 years (lane 6),15-20 years (lane 7),and 28 years (lane 8).The positions of the intact LP and LP fragments are indicated. Blots are overexposed to enhance visualization of LP fragments.
46-
possibly equivalent to an LP3-like molecule in the human. However, this LP3-like molecule in equine cartilage was never a major form, unlike LP3 in adult human cartilage. In addition, unlike the situation in human link protein, the ratio of the largest to the smaller link proteins did not change markedly with age. When immunoblots were overexposed, link protein fragments were observed, and their abundance was found to increase with the age of the animals (Fig. 6). However, w e n in the mature adult the fragments represented only a minor proportion of the total molecule.
31+
1
2
3
4
5
FIG. 5. Comparison of age-related changes in equine link protein. An equal weight of proteoglycan aggregate from animals aged 2 days (lane l),1 year (lane 2),4 years (lane 3), 15-20 years (lane 4),and 28 years (land 5) was analyzed by SDS/polyacrylamide gel electrophoresis followed by immunoblotting. The positions of molecular weight markers are indicated (Mr 46 x lo3,ovalbumin; M , 31 x lo3,carbonic an hydrase).
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Comparison of the Effects of Trypsin Treatment on Link Protein in Human, Equine, Bovine, Pig, and Rat Proteoglycan Aggregates Because little link protein similar to LP3 was detected in the mature equine and bovine cartilage, and because the human LP3 is known to be a product of proteolysis, one might presume that either
LINK PROTEIN DEGRADATION there is less proteinase activity available to act on the link protein in the nonhuman species, or that link protein from the nonhuman species is relatively resistant to proteolytic modification. As LP3 generation occurs in the N-terminal region of the link protein between the oligosaccharide attachment sites, it is possible that variation in oligosaccharide structure and size could sterically interfere with the access of proteinases. To test this possibility, human, horse, bovine, pig, and rat proteoglycan aggregates were incubated with trypsin. In all cases, link protein was completely converted to a single LP3-like form (Fig. 7), suggesting that there is no major impediment to proteinase access in the N-terminal region of the link protein molecules. The absence of a native link protein of similar mobility to the trypsin-generated link protein in the nonhuman species provides further evidence for the absence of LP3-like molecules in these species.
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determine whether this enzyme can cleave link protein from other species, the action of human cartilage stromelysin on the link protein in various proteoglycan aggregates was investigated (Fig. 8). After 24 h of incubation, human link protein was completely degraded by stromelysin to yield a single protein component that comigrates with the native LP3 component. However, the aggregates from nonhuman species, particularly in the case of the horse, showed incomplete cleavage of their link protein components. Rat link protein seemed more prone to stromelysin degradation than that of the other nonhuman species. The degraded link proteins produced by the action of stromelysin in rat, bovine, and porcine proteoglycan aggregates had mobilities comparable to that of human LP3. DISCUSSION In our study, it was shown that under electrophoretic conditions where human link protein exhibits three components, equine, bovine, and pig link protein exist as two components, and rat chondrosarcoma link protein exists as a single component. Two link protein components have also been reported in chicken xyphoid cartilage (24), canine articular cartilage (lo), and rabbit articular cartilage
Comparison of the Effects of Stromelysin Treatment on Link Protein in Human, Equine, Bovine, Pig, and Rat Proteoglycan Aggregates Previous evidence suggests that human LP3 (particularly in the neonate) is predominately a degradation product due to the action of stromelysin. To
FIG. 7. Susceptibility of link protein to trypsin digestion. Proteoglycan aggregates from the human, horse, cow, pig, and rat were analyzed directly ( - ) or following trypsin treatment ( + ) by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting.
Trypsin
,-
+,
Human
,-
+I
Horse
,-
+I
Bovine
,-
+ , I - +, Pig
Rat
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FIG. 8. Susceptibility of link protein to stromelysin digestion. Proteoglycan aggregates from human, horse, cow, pig, and rat were analyzed directly ( - ) or following treatment with stromelysin ( + ) by SDSipolyacrylamide gel electrophoresis and subsequent immunoblotting.
(33). In human cartilage, the link proteins have been defined as LPI, LP2, and LP3, based on their increasing electrophoretic mobility on SDSiPAGE. This electrophoretic heterogeneity is due to structural variation in oligosaccharide substitution (for LPl and LP2) and proteolytic modification (for LP3) (25). When two link protein components are observed in other species they may represent LP1 and LP2, LP2 and LP3, or even LPI and LP3, in comparison with the three components of human link protein. When a single link protein component is observed, it could theoretically be equivalent to any of the three forms of the human link protein. Thus, it would be desirable to have a standard nomenclature whereby link protein components can be defined on the basis of common structural features. An intact core protein with two N-linked oligosaccharides would be an LPl form, an intact core protein with one N-linked oligosaccharide would be an LP2 form, and proteolytic modification between the sites of oligosaccharide substitution would yield an LP3 form (30). On this system, the two equine link protein components and those in bovine (2) and pig cartilage (8) are LPl and LP2 forms, the single component of link protein in rat chondrosarcoma is an LP2 form, and LP3 forms are not commonly present in nonhuman species. The present data based on the electrophoretic mobility of native and trypsin-treated link proteins would indicate that while the N-terminal oligosaccharide substitution site is not always occupied, that at residue 41 is never free. The abundance of human cartilage LP3 and link
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protein fragments increases with the age of the individual (26). This is thought to be due to continual proteolytic modification of the link proteins within the extracellular matrix throughout life and the accumulation of these modified forms with age because of their continued interaction with other cartilage matrix components. In human cartilage, the enzymatic production of LP3 and link protein fragments seems to be related, since an increase in the abundance of LP3 is generally accompanied by an increase in the abundance of link protein fragments, though it is likely that different agents are involved. In this study the two extremes are represented by the hip and the meniscus, which show high and low link protein modification, respectively. In contrast, high amounts of LP3 and link protein fragments were never seen in the nonhuman cartilages, irrespective of age. Although an increase in the abundance of link protein fragments was observed with age in horses, the amount of fragmented link protein was considerably less than that found with human link protein. Thus, while the mechanisms controlling proteolytic modification of link protein might be similar in the different species, they occur to a much lower degree in the nonhuman species. One explanation for the low abundance of modified link proteins in the horse and cow, even in the mature age group, could be the relatively long life span of the human in comparison to the other species. Such longevity would favor the accumulation of structurally stable link protein degradation products. Indeed, there is no evidence to indicate that the proteolytically modified forms of link protein are less
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LINK PR 0TElN DEGRA D A T I 0N functional than their intact counterparts in their abi1i ty to form proteogl y can aggregates . Alternative explanations to account for the low proteolytic modification of nonhuman link protein are also possible. As proteolysis to generate LP3 forms occurs in the vicinity of the oligosaccharides, variation in oligosaccharide structure between the species might sterically hinder the access of proteinases. However, proteolytically modified link proteins equivalent to LP3 have been generated in vitro by trypsin (9) or clostripain ( 5 )digestion of the LPI and LP2 forms in the nonhuman species. This, together with our data on trypsin degradation of link protein from various species, indicates that there is no major impediment to proteinase access in the different link proteins. In contrast, species differences in susceptibility were observed with stromelysin. Whether this is related to the relatively large size of stromelysin is not known, but it is also possible that the difference could be related to the primary amino acid structure of the link protein. In general, the link protein sequence is highly conserved between species with the exception of the extreme N-terminus of the molecule, which is removed in the proteolytic generation of the LP3 forms. In the region where stromelysin cleaves, there is a unique amino acid substitution in the human sequence (Fig. 9) (27), which falls within the six amino acid sequence spanning the cleavage site thought to be recognized by proteinases (39). This could contribute to the enhanced susceptibility of human link protein to stromelysin cleavage. However, one cannot discount other alternative explanations at this time, as immunological evidence suggests that the folding of the protein in this region is complex (6). It is also possible that stromelysin from different species could show variation in substrate specificity, or that stromelysin is secreted or activated to a much greater degree in human cartilage than in other species. Regardless of the origin of the differences, the use of link protein as a con-
Human
t
1 10 2o D H L S D ~ T L D H D R ~ I H I O A E N G P H L L V
Bovine Horse
D H H S D N Y T V D H D R V I H I Q A E N G P R L L V
Pig
D H L S N N Y T L D H D R V I H I Q A E N G P R L L V
Rat
D H L S D S Y T P D Q D R V I H I Q A E N G P R L L V
D H R s D TE
L D H D
R
v
I
H I
Q A E N
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FIG. 9. N-terminal sequences of link protein from different species. Data for human (8), horse, pig (€9,cow, and rat (27) are depicted. The symbols used are underline, a potential site for N-linked oligosaccharide substitution; square, emphasis of the residue change in the human: and solid arrow, site of stromelysin cleavage.
venient indicator of proteolysis in cartilage during aging (28) does not appear to be applicable to other species. Acknowledgment: We thank the departments of Pathology at t h e Royal Victoria Hospital a n d the Montreal General Hospital for the provision of autopsy facilities, Ms. Elisa de Miguel for operation of the protein sequencer, and Ms. J a n e Wishart for preparing the figures. This work was supported b y the Arthritis Society of Canada and the
Shriners of North America.
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