Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz

Direkt zur Navigaton springen.
Direkt zum Inhalt springen.

Navigation/Suche

"Köpfe" der aktuellen Meldungen:

Nina Höller

Nariae Baik-Schneditz

Bernhard Schwaberger

Lukas Peter Mileder

Niels Hammer

Gerhard Pichler

Roland Malli

Gewählte Publikation:

SHR Neuro Krebs Kardio Lipid Stoffw Microb

Gerlza, T; Winkler, S; Atlic, A; Zankl, C; Konya, V; Kitic, N; Strutzmann, E; Knebl, K; Adage, T; Heinemann, A; Weis, R; Kungl, AJ.
Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.
Protein Eng Des Sel. 2015; 28(8):231-240 Doi: 10.1093/protein/gzv025 [OPEN ACCESS]
Web of Science PubMed FullText FullText_MUG

Co-Autor*innen der Med Uni Graz: Heinemann Akos; Konya Viktoria
Altmetrics:
Dimensions Citations:
Plum Analytics:
Scite (citation analytics):
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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Find related publications in this database (using NLM MeSH Indexing): 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
Find related publications in this database (Keywords): chemokines; flow chamber; fluorescence spectroscopy; heparan sulfate; surface plasmon resonance