Developing therapeutically useful pharmacological modulators of palmitoylation will require that they be developed within the context of well-characterized PAT/APT-related signaling systems. The successful development of potent, specific drugs in similarly complex systems suggests that development of useful drugs targeting PATs is feasible.
BackgroundAntiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4.ResultsWe immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127–602) or the extracellular domain plus the transmembrane domain (Aa 106–602). Positive binding was detected to both CKAP4126–602 and CKAP4106–602, suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4127–360 and CKAP4361–524 exhibited a fast association rate (k
on) and a slow dissociation rate (k
off), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127–524 region of the CKAP4 extracellular domain.ConclusionsWe determined that the CKAP4127–360 and CKAP4361–524 mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.
Interstitial cystitis (IC) is a chronic, painful bladder disease that affects mostly women and is frequently misdiagnosed due to lack of a non‐invasive test to detect the disease. Biomarkers for IC have been described, including antiproliferative factor (APF), a sialoglycopeptide that is detectable in the urine of 95‐97% of IC patients vs. normal controls. Validation of APF as a biomarker for IC has been hindered by the absence of robust assays to detect and measure its concentration in patient urine. In this study, we evaluated the utility of surface plasmon resonance (SPR) to detect and quantitate APF in solution through binding to its cellular receptor, cytoskeleton associated protein 4 (CKAP4). Five his‐tagged CKAP4 mutants were designed according to structural predictions in order to optimize the SPR assay. Each purified mutant was immobilized on a CM5 chip through amine‐coupling, and binding activity to APF and as‐APF (a desialylated APF analogue with full activity) was measured. Four of the CKAP4 mutants demonstrated a quantitative binding response to APF and as‐APF, albeit with altered sensitivities; moreover, as‐APF charged to control patient urine could be detected. Future efforts will focus on assay optimization to detect APF in urine from IC patients. USA MED Research ACQ Activity W81XWH‐13‐1‐0454
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