Branched Multimeric Peptides as Affinity Reagents for the Detection of α‐Klotho Protein

Abstract

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by singleshot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment

“…Using 50 nM of biotinylated peptides, Klotho was the only significantly enriched protein by K18-Biotin and the Klotho antibody but was not enriched by Pep-10-Biotin, K3A-Biotin or vehicle (Figure D). To further confirm the observed 130 kDa band is Klotho, the same silver stain was performed using lysate from a different cell line with low Klotho expression, CaSki cells. , No 130 kDa band was pulled down by K18-Biotin or the Klotho antibody from CaSki cell lysate, confirming that this band is Klotho (Figure S14). These results demonstrated the potential of K18-Biotin to selectively capture Klotho from cell lysate.…”

“…In the direct binding measurements by BLI, Pep-10-Biotin was immobilized on streptavidin tips, and serial dilutions of mKlotho were incubated with the tips to evaluate association and dissociation rates against Pep-10-Biotin. The direct dissociation constant ( K D ) of the Pep-10/mKlotho interaction was determined in this manner to be 2.3 ± 0.5 μM . In a competition BLI binding assay where in-solution Pep-10 competes against immobilized Pep-10-Biotin for mKlotho binding, we can assess competition K D values and confirm Klotho site-specific binding .…”

“…Meanwhile, mock transfection had no effect on fluorescence, as expected (Figure S17). In CaSki cells with low Klotho expression, , K18-TAMRA was incubated using a similar protocol to HK-2 cells. K18-TAMRA displayed a significantly lower fluorescence intensity in CaSki cells relative to that of Klotho-expressing HK-2 cells (Figure S18).…”

“…The direct dissociation constant (K D ) of the Pep-10/ mKlotho interaction was determined in this manner to be 2.3 ± 0.5 μM. 14 In a competition BLI binding assay where insolution Pep-10 competes against immobilized Pep-10-Biotin for mKlotho binding, we can assess competition K D values and confirm Klotho site-specific binding. 16 In brief, Pep-10-Biotin was immobilized onto the streptavidin biosensor tips.…”

“…However, Pep-10 only had micromolar binding affinity toward Klotho. Based on the work referenced above, we developed a series of branched peptides to target Klotho with 100-fold enhanced affinity relative to Pep-10 using automated fast-flow synthesis . Despite their enhanced performance, multimeric peptides have only modest binding affinity (10–100 nM) and a high molecular weight (>4 kDa) compared to traditional small molecules, potentially limiting their future applications in biological systems.…”