Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain

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 (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.

Self-Association of Purified Reconstituted ER Luminal Spacer Climp63

“…Palmitoylation was also detected in the NT (Planey et al, 2009), but the physiological role is not entirely clear. Interestingly, Climp63 is also implicated in several signaling events (Nisha Gupta et al, 2006;Sandoz and van der Goot, 2015;Chavda et al, 2017), with some mechanisms requiring plasma membrane localization.…”

Peptidomics analysis reveals changes in small urinary peptides in patients with interstitial cystitis/bladder pain syndrome

“…The gene encoding the APF peptide was found to be consistently overexpressed in IC/BPS patient bladder epithelial cells samples relative to controls as determined by Northern blot 27 . Chavda et al developed a surface plasmon resonance-based assay to detect a synthetic desialylated APF peptide analogue (as-APF) spiked into control urine, but this assay was not used to quantify the APF peptide from IC/BPS urine 30 . To our knowledge, our study is the first to directly measure the APF peptide from IC/BPS urine and to compare its abundance to control urine.…”

“…Thus, it is possible that alternative forms of the APF peptide may be present in IC/BPS patient samples, and these may prove to be more reliable as IC/BPS biomarkers than the form studied here. Indeed, prior studies have suggested that the desialylated as-APF peptide may occur naturally, and structure–activity studies found that APF peptide derivatives truncated at either the N- or C-terminus retain high biological activity 27 , 30 , 62 , 63 . Future studies may benefit from examining alternative forms of the APF peptide, or examining other molecules that may also be responsible for the antiproliferative activity found in IC/BPS urine.…”

“…Chromatographic purification of a bioactive component of urine/bladder tissue followed by mass spectrometry revealed a glycosylated nonapeptide (“APF peptide”), which appears to be a fragment of the frizzled-8 (FZD8) G protein-coupled receptor 27 . Subsequent studies confirmed that the APF peptide displays antiproliferative properties 27 , 28 , and appears to exert its effects through the CKAP4 receptor 29 , 30 . Despite convincing evidence that the antiproliferative bioactivity of urine may be used to discriminate IC/BPS patients 25 , to our knowledge no study has directly examined the urinary abundance of the APF peptide in IC/BPS patients versus healthy controls.…”

A ten-genes-based diagnostic signature for atherosclerosis

“…LYN is the src family gene, and it is revealed that the scavenger receptor CD36 could bind to the oxidized LDL, thus activating LYN by the recruitment of Na/K-ATPase-LYN complex and enhancing the development of atherosclerosis [ 40 ]. Cytoskeleton associated-protein 4 (CKAP4), originally identified as an endoplasmic reticulum resident protein, is a reversibly palmitoylated, type II transmembrane protein [ 41 ]. It has been revealed that CKAP4 exerts an suppressive effect on the α5β1 integrin recycling, while the the α5β1 integrin is closely associated with atherosclerosis and plays a key role in this disease [ 42 , 43 ].…”