Purpose

Treatment of osteoarthritis with novel injectable therapies could be improved by targetting the lesion sites in the articular cartilage. We have previously shown that degradation of Type II collagen in osteoarthritis occurs at the surface of the articular cartilage, leading to an accumulation of Type II collagen gelatin (1), a prime candidate for therapeutic targetting. Gelatinases A and B contain a catalytic domain as well as three fibronectin-like modules that form the collagen binding domain (CBD). The purpose of this study was to determine which of these modules is most important for binding to Type II collagen gelatin and to enhance the binding to this gelatin through mutations in its structure. The long-term goal is to deliver either drugs or cells (see Ref. 2) directly into the lesion sites.

Methods and Materials

Full length CBD, individual modules of CBD or mutations of these modules were expressed in E.coli shuffle cells, purified and then biotinylated before being tested for their ability to bind to denatured collagen types I and II by direct binding ELISA.

Results

CBD was found to have 10-fold higher binding (ie 10-fold lower Kd) to Type I collagen gelatin than Type II (Figure 1). Module 2 bound more tightly to gelatin than modules 1 or 3 (not shown), therefore we constructed a mutant CBD using three repeats of module 2 (222) and tested its binding . 222 had a 14-fold higher affinity for Type II collagen gelatin, with a Kd that was similar to that of is binding to Type I collagen gelatin (Figure 2).

Conclusion

The CBD mutant, protein 222, has a high binding capacity for Type II collagen gelatin and so could be used to target cells and drugs into cartilage lesions.

References

1. A. P. Hollanderet al., Journal of Clinical Investigation 96, 2859-2869 (1995)

2. J. P. Armstronget al., Nat Commun 6, 7405 (2015)