title: Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.
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.
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Tanja
authors.initials: T
authors.lastname: Gerlza
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Sophie
authors.initials: S
authors.lastname: Winkler
authors.affiliation: VTU Technology, Parkring 18, A-8074 Grambach/Graz, Austria.
authors.forename: Aid
authors.initials: A
authors.lastname: Atlic
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Zankl
authors.affiliation: Medizinische Universität Graz, Institute of Experimental and Clinical Pharmacology, Universitätsplatz 4, A-8010 Graz, Austria.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Konya
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Nikola
authors.initials: N
authors.lastname: Kitic
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Strutzmann
authors.affiliation: ProtAffin Biotechnologie AG, Reininghausstrasse 13a, A-8020 Graz, Austria.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Knebl
authors.affiliation: ProtAffin Biotechnologie AG, Reininghausstrasse 13a, A-8020 Graz, Austria.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Adage
authors.affiliation: Medizinische Universität Graz, Institute of Experimental and Clinical Pharmacology, Universitätsplatz 4, A-8010 Graz, Austria.
authors.forename: Akos
authors.initials: A
authors.lastname: Heinemann
authors.affiliation: VTU Technology, Parkring 18, A-8074 Grambach/Graz, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Weis
authors.affiliation: Karl-Franzens-University Graz, Institute of Pharmaceutical Sciences, Universitätsplatz 1, A-8010 Graz, Austria ProtAffin Biotechnologie AG, Reininghausstrasse 13a, A-8020 Graz, Austria Antagonis Biotherapeutics, Strasserhofweg 77a, A-8045 Graz, Austria andreas.kungl@uni-graz.at.
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Kungl
mesh_terms: D019943:Amino Acid Substitution; D018932:Chemokine CCL2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D006025:Glycosaminoglycans; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C020341:glucosaminoglycans
keywords: chemokines; flow chamber; fluorescence spectroscopy; heparan sulfate; surface plasmon resonance