Studies of the articular cartilage proteoglycan aggrecan in health and osteoarthritis. Evidence for molecular heterogeneity and extensive molecular changes in disease. G Rizkalla, … , E Bogoch, A R Poole J Clin Invest. 1992;90(6):2268-2277. https://doi.org/10.1172/JCI116113. Research Article Changes in the structure of the proteoglycan aggrecan (PG) of articular cartilage were determined immunochemically by RIA and gel chromatography and related to cartilage degeneration documented histologically by the Mankin grading system. Monoclonal antibodies to glycosaminoglycan epitopes were used. In all cartilages, three chondroitin sulfate (CS)- rich populations of large size were observed in addition to a smaller keratan sulfate (KS)-rich population. In grades 7-13 OA cartilages (phase II), molecules were significantly larger than the equivalent molecules of grades 2-6 (phase I). CS chain lengths remained unchanged. In most OA cartilages, a CS epitope 846 was elevated in content, this being most marked in phase II (mean: fivefold). Loss of uronic acid, KS, and hyaluronic acid were only pronounced in phase II OA because of variations in normal contents. Aggregation of PG was unchanged (50-60%) or reduced in OA cartilages, but molecules bearing epitope 846 exhibited almost complete aggregation in normal cartilages. This study provides evidence for the capacity of OA cartilage to synthesize new aggrecan molecules to replace those damaged and lost by disease- related changes. It also defines two phases of PG change in OA: an early predominantly degenerate phase I followed by a net reparative phase II accompanied by net loss of these molecules. Find the latest version: https://jci.me/116113/pdf Studies of the Articular Cartilage Proteoglycan Aggrecan in Health and Osteoarthritis Evidence for Molecular Heterogeneity and Extensive Molecular Changes in Disease Geihan Rizkalla, * Agnes Reiner, * Earl Bogoch,t and A. Robin Poole * *Joint Diseases Laboratory, Shriners Hospitalfor Crippled Children, Division ofSurgical Research, Department ofSurgery, McGill University, Montreal, Quebec, Canada, H3G IA6; and *Department of Orthopaedic Surgery, Wellesley Hospital, University of Toronto, Toronto, Ontario, Canada, M4Y IJ3 Abstract with its tensile strength and stiffness (2, 3). This is composed predominantly of type II collagen. Type XI collagen is present Changes in the structure of the proteoglycan aggrecan (PG) of within the fibril (4). On its surface, covalently attached to type articular cartilage were determined immunochemically by RIA II collagen in the nonhelical telopeptide regions, resides type and gel chromatography and related to cartilage degeneration IX collagen (5, 6). Interacting with this network, either directly documented histologically by the Mankin grading system. or indirectly, is a "mesh work" of high molecular weight hyal- Monoclonal antibodies to glycosaminoglycan epitopes were uronic acid (HA)' (7). A large proteoglycan (PG), called ag- used. In all cartilages, three chondroitin sulfate (CS) -rich popu- grecan, which on synthesis has a 2-4 X 106 Mr, binds to HA lations of large size were observed in addition to a smaller kera- through an amino-terminal GI globular domain (8). Each at- tan sulfate (KS)-rich population. In grades 7-13 OA cartilages tachment is stabilized by a single link protein (9, 10) that (phase II), molecules were significantly larger than the equiva- shares sequence homology with the G 1 domain (1 1, 12). lent molecules of grades 2-6 (phase I). CS chain lengths re- The core protein of aggrecan contains a second globular mained unchanged. In most OA cartilages, a CS epitope 846 domain called G2 which has considerable homology with GI was elevated in content, this being most marked in phase II ( 12): its function is unknown. Between G2 and another globu- (mean: fivefold). Loss of uronic acid, KS, and hyaluronic acid lar domain called G3 (12, 13) situated at the carboxyl-ter- were only pronounced in phase II OA because of variations in minal, there are the glycosaminoglycan attachment regions normal contents. Aggregation of PG was unchanged (50-60%) ( 12, 13). Adjacent to G2 there is a keratan sulfate (KS) attach- or reduced in OA cartilages, but molecules bearing epitope 846 ment region composed of repeating hexapeptide motif and exhibited almost complete aggregation in normal cartilages. containing up to 30 KS chains ( 13, 14). Between this region This study provides evidence for the capacity of OA cartilage to and G3 there is a high concentration of up to 100 CS chains synthesize new aggrecan molecules to replace those damaged ( 13 ). There is evidence from studies of human aggrecan, based and lost by disease-related changes. It also defines two phases on core protein structure, that this region may be composed of of PG change in OA: an early predominantly degenerate phase two subdomains called CS-I and CS-2 (13). The G3 domain I followed by a net reparative phase II accompanied by net loss has been shown to have lectin-like properties, since it binds to .of these molecules. (J. Clin. Invest. 1992. 90:2268-2277.) Key galactosyl residues ( 15). This property may permit interac- words: cartilage - proteoglycan* osteoarthritis * aggrecan * im- tions with molecules in the extracellular matrix. munochemistry Antibodies have been prepared to unsaturated sulfated and unsulfated disaccharides of chondroitin sulfate (CS), some of Introduction which remain as stubs attached to the core protein of aggrecan after eliminative of CS These antibodies Osteoarthritis is a degenerative joint disease that involves im- digestion ( 16, 17). pairment and eventually loss of joint function. It is character- react preferentially with the "stubs" that remain attached to ized by extensive of the articular in core protein, and are sulfation specific (disaccharide chondroi- degeneration cartilages tin 4-sulfate stubs attached to core both large and small joints, usually in the presence of low level protein [ AdiC4S-s], disac- charide chondroitin 6-sulfate stubs attached to core protein intraarticular inflammation (1). [AdiC6S-s], and disaccharide chondroitin stubs attached to The articular cartilage ofa diarthrodial joint is composed of core protein More antibodies to native an extensive network of collagen fibrils that [AdiCOS-s]). recently, provide cartilage CS epitopes have been prepared ( 16, 18, 19). These are usually most commonly found in fetal or embryonic tissues. The ma- Address all correspondence to Dr. A. Robin Poole, Shriners Hospital jority ofthe epitopes they recognize remain to be characterized. for Crippled Children, Division of Surgical Research, Department of Antibodies to KS (20, 21 ) have been shown to recognize highly Surgery, McGill University, 1529 Cedar Ave., Montreal, Quebec, Can- sulfated domains on this glycosaminoglycan (22). Use ofthese ada, H3G 1A6. antibodies in immunoassays permits the detection and mea- Geihan Rizkalla's current address is Department of Psychiatry, surement of PG and fragments thereof in cartilages in the pres- Royal Victoria Hospital, McGill University. ence of other macromolecules ( 17). Receivedfor publication 26 February 1992 and in revisedform 31 July 1992. 1. Abbreviations used in this paper: CS, chondroitin sulfate; AdiC4S-s, J. Clin. Invest. disaccharide chrondroitin 4-sulfate stubs attached to core protein; © The American Society for Clinical Investigation, Inc. AdiC6S-s, disaccharide chondroitin 6-sulfate stubs attached to core 0021-9738/92/12/2268/10 $2.00 protein; GuCl, guanidinium chloride; HA, hyaluronic acid; KS, kera- Volume 90, December 1992, 2268-2277 tan sulfate; OA, osteoarthritis; PG, proteoglycans; UA, uronic acid. 2268 G. Rizkalla, A. Reiner, E. Bogoch, and A. R. Poole These proteoglycans constitute up to 10% of the wet weight removal. The age range of the patients was 59-77 yr. Altogether, six of cartilage. Because of their high content of glycosaminogly- normal and seven OA joints were studied for total contents, molecular cans, they become very hydrated and can absorb up to 50 times sizes, and aggregation. their weight of water. But this hydration is constrained by the 1-cm2 specimens of the whole depth of cartilage ( 100-200 mg in tensile strength of the collagen network. This results in a swell- wet wt) from the distal weight bearing areas of the femoral condyles of ing or osmotic pressure that endows cartilage with its compres- normal and OA joints were removed, taking care to exclude underlying subchondral bone. In some of the normal and OA samples, two speci- sive stiffness and hence its reversible deformability (23-25). As mens were prepared at different sites ofthe weight-bearing region ofthe human articular cartilage ages, there is an accumulation of the same femoral condyle to study variations within different sites of the G I globular domain (26) and of smaller KS-rich aggrecan mol- same joint. Specimens were frozen sectioned at 20 Mm with a cryostat ecules (27, 28). These proteoglycan degradation products in- (Tissue TEK II; Miles Scientific Div., Miles Laboratories Inc., Naper- crease in content mainly in the deep zone (28, 29). The degra- ville, IL) before extraction. Fetal, epiphyseal, and articular cartilages dation probably occurs over a period of time in the extracellu- were obtained within 16 h (at 40C) of therapeutic abortion. lar matrix. Further evidence for extracellular degradation is Histology. A full depth sample adjacent to the specimen chosen for provided by the progressive accumulation with aging of link immunochemical and biochemical analyses was removed and fixed for protein of reduced size (30). This probably results in part from 24 h in 10% formalin in 25 mM sodium phosphate, pH 7.0, for histol- the extracellular action of proteinases, such as the metallopro- ogy. After wax embedding, 6-Am thick sections taken at two different levels perpendicular to the articular surface were cut and stained with teinase stromelysin (31 ). hematoxylin and eosin or with safranin-O and fast green-iron hema- There have been many studies of the PG aggrecan in osteo- toxylin. Sections were graded according to Mankin's histological grad- arthritic articular cartilages. These have sometimes produced ing system for OA cartilages (38).