A new immunoassay was developed to detect denaturation of type II collagen in osteoarthritis (OA). A peptide, al(II)-CBllB, located in the CB11 peptide of type II collagen, was synthesized and used to produce a monoclonal antibody (COL2-3/4m) of the IgG,(K) isotype. This reacts with a defined epitope in denatured but not native type II collagen and the a3 chain of type XI collagen. The latter is present in very small amounts (about 1% wt/wt) in cartilage relative to the al (II) chain. By using an enzyme-linked immunosorbent assay, type II collagen denaturation and total type II collagen content were determined. The epitope recognized by the antibody was resistant to cleavage by a-chymotrypsin and proteinase K which were used to extract al (II)-CB11B from the denatured (a-chymotrypsin soluble) and residual native (proteinase K soluble) collagen a-chains, respectively, present in human femoral articular cartilage. Type II collagen content was significantly reduced from a mean (range) of 14% (9.2-20.8%) of wet weight in 8 normal cartilages to 10.3% (7.4-15.0%) in 16 OA cartilages. This decrease, which may result in part from an increased hydration, was accompanied by an increase in the percent denaturation of type II collagen in OA to 6.0% of total type II collagen compared with 1.1% in normal tissue. The percent denaturation was ordinarily greater in the more superficial zone than in the deep zone of OA cartilage. (J. Clin. Invest.

Multiple and variable tyrosine sulfation in extracellular class II leucine-rich repeat proteins/proteoglycans were characterized by mass spectrometry. The sulfogroup on tyrosine is labile and is released from peptides under normal mass spectrometric conditions. Thus, special approaches must be considered in order to identify this modification. By using a combination of mass spectrometry studies operating in negative and positive ion mode, tyrosine sulfation could be identified. In positive mode, the peptides normally appeared non-sulfated, whereas in negative mode a mixture of sulfated and non-sulfated species was observed. A combination of peptides released by different proteinases was used to obtain details on the locations of sulfate groups. Multiple tyrosine sulfates were observed in the N-terminal region of fibromodulin (up to 9 sites), osteoadherin (up to 6 sites), and lumican (2 sites). Osteoadherin contains two additional sulfated tyrosine residues close to its C terminus. We also identified an error in the published sequence of bovine fibromodulin, resulting in the replacement of Thr 37 by Tyr 37 -Gly 38 , thus increasing its homology with its human counterpart.Tyrosine sulfation is a post-translational modification found on many secreted and membrane-bound proteins. As much as 1% of all tyrosine residues of the total protein in an organism can be sulfated, making this the most common post-translational modification of this residue (1). A number of proteins have been reported to contain sulfated tyrosines (2). Sulfation is suggested to occur at tyrosines located in close proximity to acidic residues (3). The existence of two different tyrosylprotein sulfotransferases, TPST-1 1 and TPST-2, might explain the diversity of sequences that are sulfated. Each enzyme may have a different substrate specificity and act on a different subset of target proteins. Tyrosine sulfation of chemokine receptor CCR-5 by TPST-1 and TPST-2 follows a discrete pattern and temporal sequence (4). The N terminus contains the sulfation sites with 0 -4 sulfogroups present. TPST-1 null mice (5) appeared healthy but had a ϳ5% lower average body weight than wild type animals. In addition, although the fertility of (Ϫ/Ϫ) males and females is normal, they have significantly smaller litters because of fetal death between 8.5 and 15.5 days postcoitum.Regulation of tyrosine sulfation in vivo by enzymatic sulfate removal is still largely unknown. However, human arylsulfatase A and B reside in the lysosome where they are thought to participate in the degradation of tyrosine sulfated proteins (6). In contrast, arylsulfatase E is found in the Golgi compartment (7). Mutations in this gene cause chondrodysplasia punctata, a congenital disorder characterized by abnormalities in cartilage and bone development (7-12).The biological role of tyrosine sulfation has been unclear, but recent studies have shown its functional importance in leukocyte adhesion (13-16), hormone synthesis (17-19), chemokine receptor signaling (20 -26), and hemostasis (27-33)....

Integrity of cartilage fails in joint disease. The current work aimed to identify candidate active proteinases in joint diseases using an in vitro model for cartilage degradation induced by interleukin-1. A critical event in the process of cartilage destruction in joint disease is the failure of the collagen fiber network to maintain integrity. Proteins binding to the surface of the fibers are likely early points of failure. Fibromodulin, a member of the leucine-rich repeat protein family, is one predominant protein in cartilage and is known for its roles in the formation of collagen fibrils and sustained interaction with these formed fibers. Cleavage removes the tyrosine sulfate-rich region in the N terminus of fibromodulin. Whereas fibromodulin bound to collagen in tissue was digested, purified fibromodulin was not cleaved. In contrast an N-terminal 10-kDa fragment, Gln 19 -Lys 98 , of the protein generated by Lys-C digestion contains the cleavage site and was a substrate cleaved by the enzyme in medium from stimulated cultures. In solution, digestion of this substrate with matrix metalloproteinase (MMP)-2, -9, -8, and -13 demonstrated that only MMP-13 was capable to efficiently cleave it. The cleavage product obtained after MMP-13 digestion was identical to that observed in cleaved fibromodulin from cartilage explant cultures stimulated with interleukin-1. MMP-13 treatment of fresh articular cartilage also produced the fragment under study. The elucidation of the enzyme responsible for such cleavage may lead to treatment modalities involving its selective inhibition for patients suffering from arthritis. The known structure of the fragments permits the generation of neo-epitope antibodies to the cleavage site, which can be used to detect ongoing cartilage degradation in patients with arthritic disease, an important adjunct in monitoring disease progression, active disease, and efficacy of treatment.

The ability of the intervertebral disc to resist compression is dependent on its high proteoglycan concentration. The disc proteoglycans are classified as aggregating or non-aggregating depending on their ability to interact with hyaluronan. The majority of the aggregating proteoglycans are derived from aggrecan, though their glycosaminoglycan substitution pattern has not been determined. In contrast, the origin of the non-aggregating proteoglycans is unclear, though it has been postulated that they are derived from aggrecan by proteolysis. The present work demonstrates that keratan sulfate (KS) in the glycosaminoglycan-binding region of disc aggrecan is confined to the KS-rich domain of the core protein and is not present in association with chondroitin sulfate (CS) in the CS1 and CS2 domains. It also shows that the non-aggregating disc proteoglycans are derived from aggrecan, with the large molecules possessing both the KS-rich and CS1 domains and the smaller molecules being generated from either the KS-rich or CS2 domain. The origin and spectrum of disc proteoglycan heterogeneity is the same in both the annulus fibrosus and nucleus pulposus.

The authors identified three matrix turnover phases, related to age and grade of degeneration. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of Type II collagen. Phase II (aging and maturation) is distinguished by a drop in synthetic activity and a reduction in denaturation of Type II collagen. Phase III (degenerative) is illustrated by an increase in Type II collagen denaturation and Type I procollagen synthesis, both related to grade of tissue degeneration.