It is important to consider that the significance of COMP may be diminished in smaller animals, for instance, mice do not have COMP in their tendons32 unlike larger animals including humans40 and bovine41. and COMP (16F12). Bound lubricin and COMP (red) were identified at the cartilage surface. A second layer was also apparent below the surface. (b) Lubricin (5C11) and Gefitinib-based PROTAC 3 fibronectin (ab32419). Bound lubricin and fibronectin (red) were predominately identified at the cartilage surface. Only when the PLA signal was thinner at the surface was signal observed just below the surface. (c) Lubricin (5C11) and collagen II (sc-7763). Bound lubricin and collagen II (red) were identified only at the surface with no PLA signal further into the tissue. PLA signal are red, DAPI staining in blue. For the 3 antibody pairs, 5 separate antibodies were used as anti-lubricin 5CII was used for 2 pairs. Negative controls were performed by omitting each primary antibody for all of the five antibodies used. (d) lubricin negative control (PA3-118), (e) COMP negative control (16F12), (f) lubricin Gefitinib-based PROTAC 3 negative control (5CII), (g) fibronectin negative control (ab32419) and (h) collagen II negative control (sc-7763). As expected the Gefitinib-based PROTAC 3 PLA of lubricin and COMP (Fig.?3a) confirmed an interaction between the two proteins at the cartilage surface. Interestingly, the PLA signal from the COMP-lubricin complex was strong up to 40?m into the cartilage, particularly intense where the surface layer was diminished. PLA signals were also detected for both the lubricin and fibronectin (Fig.?3b) and lubricin and collagen II (Fig.?3c) pairs. The latter two pairs were only identified at the cartilage surface with no protein interactions apparent deeper in the tissue. This suggests that both fibronectin and collagen II, along with COMP may be involved in the adherence of lubricin to the cartilage surface. Solid phase binding assays To verify that collagen II and fibronectin bind lubricin and to determine the lubricin binding region, solid phase binding assays were performed using a range of lubricin fragments that have been previously described28. Statistical analyses compared the binding of each recombinant lubricin fragment to bovine serum albumin (BSA) binding. Collagen II isolated from bovine cartilage bound the full length lubricin (lub) with a truncated mucin domain, the L871-1078 mucin domain construct and the N-terminal fragment L25-221 (Fig.?4b) to a similar extent. Four smaller N-terminal lubricin fragments were then tested and collagen II was found to bind only the L105-160 fragment (Fig.?4c) when compared to BSA. Human blood fibronectin bound with similar intensity to the full length-truncated mucin website lubricin and L871-1078 mucin website fragment; however, fibronectin binding was more than twice as intense to the C-terminal fragment L1078-1404 (Fig.?4d). Collectively, these results verify that lubricin binds collagen II and fibronectin and that the N-terminal of lubricin is definitely important for binding with collagen II and the C-terminal for fibronectin. Open in a separate window Number 4 Recombinant (RC) lubricin forms non-covalent complexes with collagen II and Rabbit Polyclonal to SPON2 fibronectin. (a) Representation of RC lubricin constructs. Dark blue: FLAG-tagged, indicated in mammalian 293?F cells. Light blue: N-terminal fragments GST-tagged indicated in strain Rosetta 2. (b) Connection between collagen II isolated from bovine cartilage and RC lubricin fragments, full size lubricin and BSA by solid phase binding assay. (c) Connection between collagen II isolated from bovine cartilage and RC N-terminal lubricin fragments and BSA by solid phase binding assay. (d) Connection between fibronectin isolated from human being blood and RC lubricin fragments, full size lubricin and BSA by solid phase binding assay. For those assays n?=?3 and error bars are standard deviation. N- designates the amino terminus and CC designates the carboxy terminus. The transmission peptide (1C24) is definitely shown in gray. All statistical analyses compare the binding of lubricin fragments to the binding of the BSA standard. * is defined as p??0.05, and *** is defined as p??0.001. Recognition of lubricin from digested cartilage biopsy cells To confirm the observed superficial lubricin was directly in the cartilage surface, pieces of biopsy cells were digested with matrix metalloproteinase-9 (MMP-9). Cells was digested with triggered MMP-9 as well as incubated with the pro-enzymes. MMPs efficiently break down proteins of the cartilage ECM. MMP-9 (gelatinase B) cleaves elastin, aggrecan, laminin, COMP and collagens IV, V, XI, XIV34,35. MMP-9 released lubricin from your pieces of decalcified OA patient articular cartilage biopsies into the.