Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity
| Autor: | Nikola Kitic, Andreas J. Kungl, Christina Zankl, Sophie Winkler, Akos Heinemann, Roland Weis, Aid Atlic, Tiziana Adage, Elisabeth Strutzmann, Tanja Gerlza, Kerstin Knebl, Viktoria Konya |
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| Rok vydání: | 2015 |
| Předmět: |
Glycosaminoglycan binding
Chemistry Recombinant Fusion Proteins Mutant Mutation Missense Wild type Bioengineering Ligand (biochemistry) Human serum albumin Biochemistry Fusion protein Amino Acid Substitution Mutant protein medicine Humans Protein quaternary structure Molecular Biology Chemokine CCL2 Serum Albumin Glycosaminoglycans Protein Binding Biotechnology medicine.drug |
| Zdroj: | Protein Engineering Design and Selection. 28:231-240 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzv025 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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