Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity

Protein Eng Des Sel. 2015 Aug;28(8):231-40. doi: 10.1093/protein/gzv025. Epub 2015 May 11.

Abstract

Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.

Keywords: chemokines; flow chamber; fluorescence spectroscopy; heparan sulfate; surface plasmon resonance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution*
  • Chemokine CCL2 / chemistry*
  • Chemokine CCL2 / genetics
  • Chemokine CCL2 / metabolism
  • Glycosaminoglycans / chemistry*
  • Glycosaminoglycans / metabolism
  • Humans
  • Mutation, Missense
  • Protein Binding
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Serum Albumin / chemistry*
  • Serum Albumin / genetics
  • Serum Albumin / metabolism

Substances

  • CCL2 protein, human
  • Chemokine CCL2
  • Glycosaminoglycans
  • Recombinant Fusion Proteins
  • Serum Albumin
  • glucosaminoglycans