Generation of GPI-linked CCL5 based chemokine receptor antagonists for the suppression of acute vascular damage during allograft transplantation

Abstract

Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) helps to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES has previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic long-term administration of chemokine receptor antagonists, a chemokine based reagent was designed to be integrated into endothelial surfaces of the organ just before transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)-GPI was efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.

Synthetic cell surface receptors for delivery of therapeutics and probes

“…Related recombinant GPI-linked proteins have been used to protect vasculature from immune responses to transplanted organs. [98] Because GPI-linked proteins undergo clathrin-independent endocytosis, Jurkat lymphocytes treated with a GPI-linked variant of the immunoglobulin Fc receptor FcγRIII will endocytose ligands that bind this receptor. [99] Single-chain antibodies covalently linked to lipids have been used to construct related cell surface receptors.…”

Retention in the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated by two distinct mechanisms

“…FLAG-Cfr 1169, corresponding to the reported rat 1165 mutant [10], was generated by deleting the C-terminal six amino acid residues (RELKDR). In all of the GPI-anchored mutants, the extracellular domain of Cfr (amino acids 1-1141) was fused to the signal sequence for the GPI-anchor of human [13][14][15][16] were inserted between the Cterminus of the extracellular domain and the N-terminus of the transmembrane region of mouse EpCAM (between amino acids 266 and 267).…”

“…One mutant was a FLAG-tagged Cfr without the C-terminal cytoplasmic tail (FLAG-Cfr 1169), which corresponds to the rat Cfr mutant reported to be expressed on the cell surface [10] ( Figure 1A). The other comprised the FLAG-tagged extracellular domain of Cfr fused to the wellcharacterized GPI signal peptide of human CD58 [13] (FLAG -Cfr-GPI) ( Figure 1A). We expressed these two mutants in Ba/F3 cells, and monitored their intracellular distribution.…”

SERINC as a Restriction Factor to Inhibit Viral Infectivity and the Interaction with HIV

“…In vitro studies have to develop synthetic cell surface receptors that are inserted into the cell surface for the execution of their biologic functions and have the potential of biological drugs [ 86 ]. As proteins anchored by glycosylphosphatidylinositol are incorporated in the plasma membrane, they retain native protein function [ 87 ]. The use of synthetic cell surface receptors is a better strategy than the gene transfer for the manipulation of the components of the plasmatic membrane [ 86 , 87 ].…”