We show that one mechanism by which lower LMW forms of ps20 arise is usually through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. characterised and tested ps20 preparations for three biological properties: (i) interactions with glycosaminoglycans (GAG) (ii) inhibition of cell proliferation, and (iii) transglutaminase2 (TG2) mediated crosslinking of ps20 to fibronectin, a process implicated in wound healing. We show herein that ps20 preparations contain multiple molecular forms including full-length ps20 (resolving at 27?kDa), an exon 3 truncated form (22?kDa) that lacks aa113C140, and variable amounts of a putatively cleaved lower MW (15C17?kDa) species. Untagged purified ps20 preparations containing a mixture of these forms are biologically active in significantly suppressing prostate cell proliferation. We show that one mechanism by which lower LMW forms of ps20 arise is usually through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. However, CL cleavage abrogated the conversation between ps20 and solid-phase fibronectin. Therefore, we demonstrate for the first time that LMW forms of ps20 that lack a C-terminal immunogenic epitope can arise through CL cleavage and this cleavage impairs multimerisation and potential capacity to cross-link to ECM, but not the capacity of ps20 to inhibit cell proliferation. We propose that ps20 like other WFDC proteins can become associated with GAGs and the ECM. Furthermore, we suggest post-translational processing and cleavage of ps20 is required to generate functional protein species, and TG2 mediated crosslinking and CL cleavage form components of a ps20 regulatory apparatus. test. Statistical analysis was performed with Prism version 4 (GraphPad Software, San Diego, CA). Error bars represent SE, and P 0.05 was considered statistically significant. 3.?Results 3.1. ps20 enrichment through conversation with glycosaminoglycans (GAGs) reveals multiple molecular species We predicted that ps20 would bind GAGs based on known interactions of family members [27] and explored using this conversation to initially isolate and characterise the protein. To investigate the conversation between ps20 and GAGs we initially used crude conditioned media (CM) from 293T cells transfected Bosentan Hydrate Bosentan Hydrate to express ps20. CM was directly assimilated to and eluted from a heparin-sepharose column using a NaCl gradient (Fig. 1(A)). Notably, the concentrated 0.5?ml fractions eluted by addition of increasing concentrations of NaCl revealed for the first time numerous ps20 protein species of different molecular weights (MW) ranging from 27?kDa to 15?kDa. Fig. 1(B) shows the elution profile of ps20 using an ELISA to detect ps20 in the eluted fractions. The majority of bound ps20 was clearly eluted between 0.25 and 0.35?M NaCl. We then investigated the conversation between ps20 and physiologically relevant GAGs. Unlike heparin, heparan-sulphate and chondroitin-sulphate are expressed on the surface of cells and in the extracellular matrix (ECM) and have been shown to have important functions in the regulation of surface signalling and protein trafficking. Interestingly, rps20293F had notably higher affinity for heparin-sulphate than chondroitin-sulphate-A, while no specific binding to chondroitin-sulphate-C was observed at the concentrations tested. Next, to explore if ps20 can interact with cell associated GAGs IL10 we used a purified C-terminal tagged recombinant ps20 (rps20V5) probed with an antibody to the V5 tag. Notably, this tagged rps20V5 had a similar elution profile to untagged ps20 when assimilated and Bosentan Hydrate eluted using heparin-sepharose (Fig. 1(B)). 293T Bosentan Hydrate cells were treated with rps20V5 in the presence.