Abstract
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 (kon) and a slow dissociation rate (koff), 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.
Highlights
Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease
Since TM proteins that function as receptors traditionally have ligand binding site(s) located within their extracellular domain, we engineered a recombinant cytoskeleton associated-protein 4 (CKAP4) protein encompassing only the extracellular domain (Aa 127–602) tagged at the C-terminus with a His-tag (CKAP4127–602) and the extracellular domain plus the TM domain (CKAP4106– 602) with N-terminal His-tag to measure binding to APF/as-APF
To rule out the possibility that the fixed orientation of CKAP4 on the nitrilotriacetic acid (NTA) chip due to immobilization through the His-tag could account for the lack of binding, we used an alternate strategy immobilizing APF or as-APF to a CM5 chip as the ligand to test their binding to the CKAP4 extracellular domain (ED) mutants. pH scouting revealed that APF had limited/insufficient immobilization to the CM5 chip, whereas as-APF and control APF were efficiently immobilized
Summary
Antiproliferative 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); the biophysical interaction of APF with CKAP4 has not been characterized previously. It was shown that changes in MMP2, p53 and CCN2 protein expression and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all abrogated following CKAP4 siRNA knockdown in T24 bladder carcinoma cells [10, 11]. These data indicate that CKAP4 is essential for mediating the signaling and antiproliferative activity of APF
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