Abstract
Osteoarthritis (OA) is now becoming the main disease that affects public health. There is no specific medicine used for OA in clinical application until now. Recently, several studies demonstrated that OA is closely related to the complement system, and some complement regulators such as N-terminal non-collagenous domain 4 (NC4) aimed at alleviating OA have shown a promising therapeutic effect. However, targeting ability is the main limitation for NC4. In this study, a fusion protein named heparin-binding domain-N-terminal non-collagenous domain 4 (HB-NC4) was proposed to solve this problem, which could provide a better way for OA treatment. First, HB-NC4 plasmid was constructed using ClonExpress II one-step ligation kit method. And Escherichia coli BL21 was utilized to express the fusion protein, Ni2+-sepharose, and a desalting gravity column were introduced to purify HB-NC4. The results showed that 0.84 mg HB-NC4 could be obtained from a 1 L culture medium with a purity higher than 92.6%. Then, the hemolytic assay was introduced to validate the anti-complement activity of HB-NC4; these results demonstrated that both HB-NC4 and NC4 had a similar anti-complement activity, which indicated that heparin-binding (HB) did not affect the NC4 structure. Targeting ability was investigated in vivo. HB-NC4 showed a higher affinity to cartilage tissue than NC4, which could prolong the retention time in cartilage. Finally, the destabilization of the medial meniscus (DMM) model was applied to investigate HB-NC4 pharmacodynamics in vivo. The results indicated that HB-NC4 significantly slowed cartilage degradation during the OA process. In summary, compared with NC4, HB-NC4 had better-targeting ability which could improve its therapeutic effect and prolonged its action time. It could be used as a new complement regulator for the treatment of OA in the future.
Highlights
Osteoarthritis (OA) is a kind of degenerative disease that is mainly caused by joint trauma, chronic injury, or overuse of the knee
In order to increase the targeting ability and retention time of non-collagenous domain 4 (NC4), HB was fused to the N-terminal (Kalchishkova et al, 2011)
The expression and purification of heparin-binding domain-N-terminal noncollagenous domain 4 (HB-NC4) were reported for the first time
Summary
Osteoarthritis (OA) is a kind of degenerative disease that is mainly caused by joint trauma, chronic injury, or overuse of the knee. Complement activation final product, membrane attack complex (MAC, C5b-9), can form on the cell and directly cause the chondrocyte death by osmotic flux (Heinen et al, 2009). Several anti-inflammatory protein inhibitors have shown promising potentials in alleviating OA on animal models level (Ru and Robert, 2015; Michelfelder et al, 2018), such as insulinlike growth factor 1 (IGF-1) and the N-terminal non-collagenous domain 4 (NC4) (Schmidt et al, 2006; Kalchishkova et al, 2011), which could directly inhibit the classical pathway of complement. How to make therapeutic proteins target cartilage with a longer retention time through intra-articular injection is the key to the clinical application (Farach-Carson et al, 2005; Evans et al, 2011)
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