Fibronectin Decreased Its Inhibiting Activity To ADAMTS-4by Citrullination

Objective The erosion of joint cartilage largely depends on the destruction of the extracellular matrix via degradation of the proteoglycan aggrecan. Aggrecanases, which promote the degradation of aggrecan by cleaving aggrecan at the Glu373-Ala374site within the interglobular domain of the aggrecan proteinwhich, belong to the adamalysin subfamily of metalloproteinases. Aggrecanase-1(a disintegrin and metalloproteinase with thrombospondin motifs-4, or ADAMTS4) and aggrecanase-2(ADAMTS5) have been identified in cartilage and are thought to play a pivotal role in degradation of cartilage aggrecan. Aggrecanases have been identified in the synovial fluid of RA and osteoarthritis (OA) patients and the level of it have been found higher in RA than in OA. Aggrecanases have been found to play a role in the process of cartilage degradation. Some researches have found that the proteolytic activity of ADAMTS4is inhibited by fibronectin (FN) through interaction with their C-terminal domains and the aggrecanase activity of ADAMTS4was dose-dependently inhibited by FN, and it is suggested that the aggrecanase activity of ADAMTS4is inhibited by FN through the interaction of their C-terminal domains and that the extracellular regulatory mechanism of ADAMTS4activity is involved in the degradation of aggrecan in arthritic cartilage. PADI4is a member of the peptidylarginine deaminase family. This enzyme converts post-translational peptidylarginine to citrulline, termed citrullination. Following citrullination, many proteins change their conformation and functional activity. The objective of the present study was to observe the inhibiting activities of fibronectin(FN) and citrullinated fibronectin(cFN) to ADAMTS-4, then to explore the extracellular regulative mechanisms of ADAMTS-4.Materials and Methods The heparin-binding40-kDa fragment of human plasma fibronectin was bought from Millipore company,which was purified from a chymotryptic digest of human plasma fibronectin. This preparation contains the CS-1heparin-binding region of fibronectin and has high connecting activity to FN fragments. PADI4from rabbit skeletal muscle was bought from Sigma company. The full-length93kDa recombinant human ADAMTS4protein was prepared by the technical service of Origene,A short version of the ADAMTS4(53kDa) recombinant protein that started at the catalytic domain and ended before the spacer domain (Phe213-Cys685) was commercially obtained from R&D. Anti-ADAMTS4antibodies against the enzyme’s N-terminus, propetide domain and C-terminus, and the anti-citrulline antibody were bought form Abcam company.We got an aggrecanase activity kit from Abnova company to test the proteolytic activity of ADAMTS4. This kit measure the proteolytic activity by comparing the ARGSVIL peptide between different aggrecanases.The peptide fragments were produced by digestion with aggrecanase.Fibronectin was incubated with PAD in order to transfer FN into cFN and Citrullination was verified by western blot analysis. Enzyme-linked immunosorbent assay (ELISA) was applied to compare the activity of binding to different kinds of ADAMTS-4between FN and cFN.The proteolytic activity of the two kinds of ADAMTS-4after incubation with FN or cFN was determined using an aggrecanase activity kit. Statistical analysis of the data was performed using the SPSS V.19.0software package (SPSS, USA). The t test with different samples was adopted for comparison between groups, The Comparisons between groups were tested by one-way ANOVA analysis and LSD test. P-values of less than0.05were considered to be significant.Results 1. Western blot analysis revealed that the heparin-binding40-kDa fragment of human plasma fibronectin was modified to citrullinated fibronectin when treated with rabbit PADI4.2. The absorbance at405nm of citrullinated FN (0.624±0.033) was detected lower than uncitrullinated FN (2.182±0.042)(t=2.388, P=9.186E-07) when both of them incubated with full-length ADAMTS-4protein. Additionally, the recombinant ADAMTS4protein with a truncation at the C-terminus displayed low absorbance at405nm when the enzyme was incubated with both FN (0.934±0.012) and cFN (0.971±0.024),(t=2.388, P=0.075)3. Large amounts of ARGxx peptide were detected with full-length ADAMTS-4in aggrecanase activity assay(0.908±0.088nmol/L), but significantly less when in the presence of FN and ADAMTS-4(0.573±0.000nmol/L,t=6.594, P=0.003). The production of this peptide was more when full-length ADAMTS-4was incubated with cFN(0.830±0.020nM,t=22.257, P=2.413E-05) than with FN.4. The reaction containing the truncated ADAMTS4without FN or cFN yielded the highest concentration of ARGSVIL peptide(36.420±3.673nM), peptide production was not significantly altered when FN (41.099±0.101nM, P=0.092) or cFN (41.064±0.083nM, P=0.0094) were added to the reaction.Conclusions1. heparin-binding40-kDa fibronectin fragment could be modified to citrullinated fibronectin when treated with rabbit PADI4.2. FN could bind to the90kDa ADAMTS-4and decrease the proteolytic activity of the enzyme significantly. The binding affinity of ADAMTS-4to citrullinated FN was significantly decreased compared with its binding affinity to uncitrullinated FN, and this consequently releases the proteolytic activity of the enzyme to degrade aggrecan.3. A truncated ADAMTS4recombinant protein that lacked the C-terminus was exerts strong activity of cleaving aggrecan at the Glu373-Ala374site within the interglobular domain of the aggrecan protein,and this activity will not be changed neither by bind to FN nor cFN.