ABSTRACT
The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP-1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester-peptide segment was synthesized using the sulfonamide safety-catch linker and 9-fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full-length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP-1 cells, as expected. CCL2 was crystallized and the structure was determined by X-ray diffraction at 1.9-A resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9-Thr10-Cys11, which form an intersubunit antiparallel beta-sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety-catch linker and NCL.
Subject(s)
Chemokine CCL2/chemistry , Chemokine CCL2/chemical synthesis , Protein Structure, Quaternary , Protein Structure, Tertiary , Amino Acid Sequence , Chemokine CCL2/genetics , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Folding , Protein Multimerization , Radioligand AssayABSTRACT
Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.
Subject(s)
Chemokine CCL2/pharmacology , Chemotaxis, Leukocyte/drug effects , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Myelin Sheath/drug effects , Adult , Animals , Cell Proliferation/drug effects , Chemokine CCL2/chemical synthesis , Chemokine CCL2/therapeutic use , Chemotaxis, Leukocyte/immunology , Disease Models, Animal , Dose-Response Relationship, Drug , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Female , Humans , Immunologic Factors/pharmacology , Immunologic Factors/therapeutic use , Interferon-gamma/metabolism , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Myelin Sheath/immunology , Myelin Sheath/pathology , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Wallerian Degeneration/drug therapy , Wallerian Degeneration/immunology , Wallerian Degeneration/physiopathologyABSTRACT
Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.
Subject(s)
Chemokine CCL2/chemical synthesis , Chemokine CCL2/pharmacology , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Binding, Competitive , Calcium/metabolism , Cell Line , Chemokine CCL2/chemistry , Chemotaxis/drug effects , Chemotaxis/physiology , Humans , Ligands , Molecular Sequence Data , Molecular Weight , Protein Binding , Receptors, CCR2 , Receptors, Chemokine/drug effects , Structure-Activity RelationshipABSTRACT
Human monocyte chemoattractant protein 1 (MCP-1, CCL2) is a 8.6-kDa protein that has been implicated in a number of diseases including atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease and cancer. As part of a program to identify antibodies against MCP-1, we synthesized site-specific, biotinylated human MCP-1 analogs to be used for panning of an antibody phage display library. In contrast to material obtained from random biotinylation, the site-specific biotinylated analogs were homogeneous and retained full activity.
Subject(s)
Chemokine CCL2/chemistry , Amino Acid Sequence , Biotinylation , Calcium/metabolism , Cell Line , Chemokine CCL2/chemical synthesis , Chemokine CCL2/pharmacology , Humans , Molecular Sequence Data , Molecular Weight , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationSubject(s)
Chemokines, CC/chemical synthesis , Chemokines, CC/isolation & purification , Chemokines, CXC/chemical synthesis , Chemokines, CXC/isolation & purification , Intercellular Signaling Peptides and Proteins , Protein Folding , Chemokine CCL2/chemical synthesis , Chemokine CCL2/isolation & purification , Chemokine CXCL12 , Chemokine CXCL9 , Chemokines/chemical synthesis , Chemokines/isolation & purification , Chemokines, CC/metabolism , Chemokines, CXC/metabolism , Chromatography, High Pressure Liquid , HumansABSTRACT
The affinity-based N (alpha)-amino protecting group tetrabenzo[a,c,g,i]fluorenyl-17 methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-containing protein in the reduced state. Oxidative folding was then used to furnish the biologically active beta-chemokine MCP-1.
