Structure-Activity Analysis of Cathepsin K/Chondroitin 4-Sulfate Interactions

Abstract

In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wildtype catK⅐C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.Collagen degradation is a natural process observed in bone remodeling, wound healing, and organ development. However, excessive collagen degradation is implicated in many serious diseases such as osteoporosis, different forms of arthritis, and some vascular disorders. Triple-helical collagens are the major organic components of bone matrix (type I collagen) and of cartilage (type II collagen). These collagens are highly resistant to general proteolysis, and it requires specific peptidases for their degradation. Cathepsin K (catK), 7 a member of the papain family of lysosomal cysteine peptidases, is the predominant peptidase of bone-degrading osteoclasts (1, 2). It has the ability, unique among mammalian proteinases, of cleaving triple-helical collagen at multiple sites (3, 4). Analysis of catK activities revealed that the collagen-degrading activity is not proportional to the general peptidase activity suggesting some other factors affect the specificity of catK. It was shown (5, 6) that the ability to cleave collagen is highly dependent on the formation of an oligomeric complex of catK with glycosaminoglycans, in particular, with chondroitin 4-sulfate (C4-S). Glycosaminoglycans are naturally present in bone and cartilage in sufficient quantities to form complexes with catK. Complex formation with glycosaminoglycans is unique for catK among other papain-like proteases (6, 7). It is remarkable that monomeric catK has no significant collag...

Structural basis of collagen fiber degradation by cathepsin K

“…The structures have been refined to 2.62 Å (DS) and 2.02 Å (C4-S) with data collection and refinement statistics summarized in Table S1. The overall structure of CatK in both complexes is highly similar to previously published structures (18,20). Superposing these structures on CatK-E64 C4-S structure [Protein Data Bank (PDB) ID code 3C9E] shows a CatK rmsd of (0.373) for the DS and (0.311) for the C4-S complex.…”

“…The C4-S and DS GAGs in our structures both reveal a linear orientation of the sugar chain in contrast with the previously published cosine-curved shape of C4-S (18,20). We speculate that the conformation of the GAG chain is determined by adopting an optimal fit into the binding site it docks into, as has been previously proposed (22).…”

“…This dimer is C-shaped with the GAG chain at its center and apposed to the active site cleft of CatK molecule B. Here, two CatK molecules bind to each other in a head-to-tail organization, whereas in the previously solved CatK-GAG complex a dimer formation revealed a head-to-head organization (20). To postulate a mechanism of collagen digestion, we built a CatK dimer based on unique contacts with a single GAG chain and docked a tropocollagen into its central groove (Fig.…”

Chondroitin Sulfate Promotes Activation of Cathepsin K

“…Numerous in vitro studies have demonstrated that activated CatK inefficiently degrades collagen in solution, requiring several hours for ϳ2 M of activated CatK at the optimal acidic pH to digest just a few micromolar concentration of collagen type I (2,32,33). This apparently weak collagenase activity of CatK in vitro is not consistent with the efficient removal of bulk collagen during osteoclastic bone resorption, suggesting that other factors may enhance the collagenase activity of CatK.…”

Proteases and Cartilage Degradation in Osteoarthritis

“…Unlike the MMPs, cathepsins are single domain proteases which do not rely on additional modules to bind to their extracellular matrix substrates (Turk et al, 2001). However, the collagenolytic activity of cathepsin K is dependent on the presence of chondroitin 4-sulfate CS (Li et al, 2000) a major component of the aggrecan molecule which forms well-defined complexes with the enzyme (Cherney et al, 2011). While it was originally assumed that cathepsin K is unique to the osteoclast (and this cell does indeed contain huge amounts of the protease), many other cell types are now known to produce the enzyme (Anway et al, 2004;Sukhova et al, 1998).…”