1. Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage 
J.S. Mort, F. Beaudry, K. Théroux, A.A. Emmott, H. Richard, W.D. Fisher, E.R. Lee, A.R. Poole, S. Laverty 
Osteoarthritis and Cartilage 
Volume 24, Issue 8, Pages (August 2016)
DOI: /j.joca
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2. Fig. 1
Post-translational modification of equine type II collagen and its cleavage by cathepsin K. (A) Sequence of the equine collagen α1(II) chain beginning at the pepsin cleavage site in the N-telopeptide40 and including the triple-helical region followed by the C-telopeptide. Residue numbering represents the complete mature collagen sequence (Uniprot entry Q28396)41 starting at the N-telopeptide. Proline residues identified as hydroxylated are indicated as O, and glycated lysine residues are underlined. The cathepsin K cleavage positions, indicated by triangular arrowheads, are based on the tryptic fragments outlined in white (N-terminal fragment) and black (C-terminal fragment) with the associated mass and charge. The P1 residues are indicated in bold. Also indicated is the previously determined cathepsin K cleavage position (open arrow)13 and the classical collagenase ¾/¼ site (MMP). Peptide sequences used for antibody production (C2K77 and C2K80) are shown adjacent to the corresponding cleavage sites. (B) SDS/PAGE gradient gel analysis of equine type II collagen products following cathepsin K cleavage for 2 h using increasing enzyme concentrations. The cathepsin K band is indicated on the right. The migration positions of the pre-stained standards are indicated on the left for reference. However, as is well established, the anomalous migration of collagen α chains precludes correlative calculation of the molecular size of the fragments42,43. (C) Alignment of the sequences of human and equine type II collagen in the region of the C2K77 neoepitope and the sequence used to produce the antibody to the equivalent human epitope.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
HPLC-MS characterization of tryptic peptides of cathepsin K digested equine type II collagen identifying the most N-terminal cathepsin K generated cleavage site. HPLC-MS/MS spectrum of the proteolytic fragments (A) KPGKSGERGPOGPQGAR [190–206] at m/z 847 [M+2H]2+ and (B) GPOGPOGKPGDDGEAG [174–189] at m/z 719 [M+2H]2+.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
WebLogo representation of the cathepsin K cleavage site sequences in equine type II collagen. The height of each letter is dependent on the sequence conservation at each subsite and is obtained by multiplying the frequency of that residue at that position by the total information content at that position. Arrow shows the site of cleavage. Hydroxyproline is taken as proline and is the predominant proline form found at the P1 position.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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5. Fig. 4
Generation of type II collagen cleavage products by cathepsin K. (A) Equine and (B) human collagen were digested with cathepsin K at the concentrations indicated (pH 5.5, 32°C) and the products analyzed by SDS/PAGE and immunoblotting. The lanes show protein staining (CBB) and immunostaining for the two most N-terminal cleavage products indicated in Fig. 1(A).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Fig. 5
Immunoassay of human collagen cleavage product C2K77. (A) Competitive binding assay for the immunizing peptide. (B) Assay of immunoreactive products of human type II collagen (0.4 mg/mL) digested (pH 5.5, 32°C) for 2 h with the cathepsin K concentration indicated. Values show means and 95% confidence limits of triplicate determinations.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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7. Fig. 6
Osteochondral sections of the distant metacarpus from a horse with OA. (A) Safranin O fast green stained section. The pink stain reflects proteoglycan content of the cartilage. A landscape of OA pathology is present in the section (Modified Mankin osteoarthritic grade 1014). There is a loss of Safranin O staining at the surface of the articular cartilage but the structure is intact to the left of the image (arrow head). Moving from left to right there is a complete loss of the Safranin O staining and progressive erosion of the hyaline articular cartilage to the calcified cartilage. There is a complete erosion of the hyaline articular cartilage to the calcified cartilage on right of image (arrow). (B) Immunostaining of the corresponding section to A with the novel equine specific C2K77 polyclonal antibody raised in the rabbit that detects cleavage of type II collagen by cathepsin K. The increased brown staining reveals cathepsin K cleavage in the hyaline cartilage co-localized to areas of proteoglycan loss. (C, D & E) Images of control sections from equivalent regions to that highlighted in (B). (C) Pre-immune serum from the same rabbit prior to immunization with the peptide to produce the antibody C2K77. (D) Absorption control and (E) the dilution buffer control. (Bar = 1 mm).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. 7
Localization of collagen cleavage products in human osteoarthritic cartilage. Cathepsin K-generated type II collagen cleavage CK277 epitope staining in articular cartilage removed from knees of two arthroplasty patients (A and B) showing pericellular staining particularly towards the surface of the cartilage. The surface of the cartilage is fibrillated with loss and disorganization of the matrix. In contrast, there was a lack of staining in the normal appearing cartilage remote from the lesion (C) or in the lesional area when normal IgG was substituted for immune globulin (D). (Bar = 100 μm).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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