ABSTRACT
Bioactive peptide drugs hold promise for therapeutic application due to their high potency and selectivity but display short plasma half-life. Examination of selected naturally occurring peptide hormones derived from proteolytic cleavage of the proopiomelanocortin (POMC) precursor lead to the identification of significant plasma-stabilizing properties of a 12-amino acid serine-rich orphan sequence NSSSSGSSGAGQ in human γ3-melanocyte-stimulating hormone (MSH) that is homologous to previously discovered NSn GGH (n = 4-24) sequences in owls. Notably, transfer of this sequence to des-acetyl-α-MSH and the therapeutically relevant peptide hormones neurotensin and glucagon-like peptide-1 likewise enhance their plasma stability without affecting receptor signaling. The stabilizing effect of the sequence module is independent of plasma components, suggesting a direct effect in cis. This natural sequence module may provide a possible strategy to enhance plasma stability, complementing existing methods of chemical modification.
Subject(s)
Glucagon-Like Peptide-1 Receptor/chemistry , Melanocyte-Stimulating Hormones/chemistry , Membrane Proteins/chemistry , Pro-Opiomelanocortin/chemistry , Receptor, Melanocortin, Type 1/chemistry , Amino Acid Sequence , Cyclic AMP/metabolism , Gene Expression , Glucagon-Like Peptide-1 Receptor/blood , Glucagon-Like Peptide-1 Receptor/genetics , HEK293 Cells , Humans , Melanocyte-Stimulating Hormones/blood , Melanocyte-Stimulating Hormones/genetics , Membrane Proteins/blood , Membrane Proteins/genetics , Peptides/blood , Peptides/chemical synthesis , Pro-Opiomelanocortin/blood , Pro-Opiomelanocortin/genetics , Protein Isoforms/blood , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Stability , Receptor, Melanocortin, Type 1/blood , Receptor, Melanocortin, Type 1/genetics , Receptors, Neurotensin/blood , Receptors, Neurotensin/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.
