Synthesis of a Ganglioside GM1 Anchor for Membrane Tethered Ligands of G-Protein Coupled Receptors.
Sidhom, Eriene-Heidi.
2013
- G-protein coupled receptors, as a class of proteins, are most important therapeutically. It is estimated that over 50% of prescription drugs target these receptors.1 At nearly 1000 proteins, this family is divided into 5 classes, or families. The largest of these is Class A, the Rhodopsin family. This class is identifiable by conserved sequences which are critical for membrane activation. Among ... read morethese receptors is Chemokine-like Receptor 1 (CMKLR1 or Chem32), which is most highly expressed on leukocytes. Its natural ligand, chemerin, is shown to have chemoattractant capabilities and other pro-inflammatory characteristics.26 However, therapeutically promising are the short C-terminal peptides, naturally derived from the bioactivation of chemerin, which show potent antagonist and anti-inflammatory properties.7 Learning more about this receptor could be important, especially for its therapeutic implications. Classically, the targeting of GPCRs has been done through soluble ligands which bind to extracellular orthosteric or allosteric binding sites. However, in 2002, a new method of targeting receptors, termed pepducins, was presented by Covic et al.8,9 They targeted various Class A GPCRs by taking advantage of the critical third inner loop which shifts significantly during activation due to movement of the sixth transmembrane domain.1012 The method was reliant on the usage of palmitic acid to tether the peptide to the plasma membrane and localize it specifically to the cytoplasmic leaflet. This concept of membrane-tethered ligands was applied to the extracellular leaflet in 2009 by Fortin et al., in which they targeted Class B GPCRs using peptide anchors mimicking the transmembrane domain of GPCRs to ensure accurate polarity.13 They later moved to a lipid anchor, specifically GM1.14 Here, we present the synthesis and purification of a maleimide functionlized GM1 (mGM1) on the terminal sialic acid from natural GM1. Additionally, the synthesis and purification of a biostable chemerin peptide (sChem9-PEG8-MPA), linked to a polyethylene glycol polymer (PEG8) and functionalized with a thiol, to be reactive with the maleimde on GM1. Future experiments will need to optimize the reaction and purification of the mGM1 and sChem9-PEG8-MPA. Following successful purification of the compound, cell experiments using a luciferase assay and cells expressing CMKLR1 can determine the efficacy of the GM1-tethered ligand versus soluble ligands. If this method can be generalized to targeting other GPCR receptors it could potentially be a valuable therapeutic and investigative tool.read less
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