Chondroitin sulphate proteoglycans (CSPGs) are ubiquitous molecules present within virtually all tissues, and undertake many roles from tissue stabilization and hydration, to collagen fibrillogenesis. Members of the hyaluronidase (HYAL) family cleave not only hyaluronan but also chondroitin sulphate (CS). HYAL4 degrades CS, but not hyaluronan and is present in the placenta, skeletal muscle and testis. Here, we were interested in determining whether HYAL4 was produced by mast cells and to investigate the CS structures generated following HYAL4 digestion.
Mast cells were identified in human skin by histochemical stains such as Leder stain, toluidine blue and immunohistochemically with an anti-tryptase antibody. HYAL4 was shown to be present within cells of similar morphology and localisation to those characterised as mast cells, which was confirmed by flow cytometry and Western blot analyses. Interestingly, flow cytometry analysis of mast cells revealed the presence of CS structures including the CS neo-epitope detected with the monoclonal antibody 2B6, which is well characterized for its ability to bind the 4-sulphated CS stub epitope after digestion with the bacterial lyase, chondroitinase ABC. Generation of this neo-epitope was further investigated by digestion of CSPG isolated from bovine nasal cartilage, as well as isolated CS-A and CS-C chains with HYAL4. Degradation of CS chains by HYAL4 demonstrated the generation of neo-epitopes detected with 2B6 and a similar antibody 3B3 antibody that binds to the 6-sulphated CS stub epitope.
This work shows for the first time that mast cells synthesise HYAL4 and that it cleaves CS to generate the 2B6 and 3B3 epitopes. These data support the idea that mast cells may alter the structure of chondrotin sulphate in response to their microenvironment, which may have important ramifications in the regulation of collagen fibrillogenesis and tissue remodelling, supporting the hypothesis that inflammatory cells may have a regulatory role in these events.