Modulation of Human CXCL12 Binding Properties to Glycosaminoglycans To Enhance Chemotactic Gradients

Abstract

Controlled release of active biomolecules is an attractive approach to modulate chemotactic gradients and accordingly the recruitment of cells, e.g. endothelial progenitor cells to improve wound healing or stimulate angiogenesis after myocardial infarction. Here, we developed variants of hCXCL12, also named stromal cell-derived factor 1α, a chemokine that activates the CXCR4 and consequently recruits tissue specific stem and progenitor cells. hCXCL12 variants were designed to bind to glycosaminoglycans (GAGs) with different affinities in order to modulate its release. Sixteen analogs were recombinantly produced, characterized, and tested for their GAG-binding property. The most promising variants hCXCL12 K24/K27/R41/R47A and hCXCL12 Q48K were used for release studies from starPEG-heparin-hydrogels. The reduced GAG affinity led to a fast release of hCXCL12 K24/K27/R41/R47A, whereas hCXCL12 Q48K was slowly released over 2 weeks due to its increased binding strength compared to wild type hCXCL12. Migration of Jurkat cells and early endothelial progenitor cells was proven to demonstrate the applicability of the approach to endogenously CXCR4 expressing cell types. Thus, this work offers new options for enhancing chemotactic hCXCL12 gradients by a combination of native and modified hCXCL12 variants to improve and prolong the recruitment of CXCR4-positive stem and progenitor cells to injured sites.

Peptide‐mediated surface coatings for the release of wound‐healing cytokines

“…This will have many benefits, as synthetic compounds provide higher structural control and allow fine‐tuning of the protein binding affinity. One might imagine the creation of a more stable gradient by modulating the strength of the affinity interactions (Vulic & Shoichet, 2014), either the GAG component with higher or lower protein‐binding affinities or engineered proteins displaying a stronger or weaker GAG‐binding affinity (Spiller et al, 2019). In both cases, mussel‐derived surface‐binding peptides provide a versatile platform that is additionally modifiable with other bioactive peptides.…”

“…Full‐length CXCL12, carrying a C‐terminal methionine due to Escherichia coli expression, was produced, refolded, and purified as described previously (Spiller et al, 2019). Peptides were immobilized and subsequently loaded with 1 μM heparin (M W = 15 kDa, DS = 1.8–2.2) for 1 h and 1 μM CXCL12 for 2 h. After another washing step with TBS‐T, wells were blocked with 10% BSA in TBS and bound CXCL12 was detected using mouse‐anti‐SDF‐1 (1:500 in 1% BSA in TBS) and goat‐anti‐mouse‐IgG‐horseradish peroxidase (HRP; 1:5,000 in TBS).…”

“…Full-length CXCL12, carrying a C-terminal methionine due to Escherichia coli expression, was produced, refolded, and purified as described previously (Spiller et al, 2019…”

Protease-Triggered Release of Stabilized CXCL12 from Coated Scaffolds in an Ex Vivo Wound Model

“…Diverse immobilization strategies for growth factors and cytokines have been greatly reviewed previously [74,75]. Heparin-based PEG hydrogels loaded with CXCL12 are often applied [76][77][78], and hydrogels packed with CXCL12 mutants have been proven to attract early endothelial progenitor cells with a long-term release profile upon mutations of the heparin binding site of CXCL12 [26,79]. Other non-covalent approaches used a heparin-based sandwich-like setup, where the chemokine is bound by soluble heparin, which in turn is immobilized by a heparin-binding peptide [30,80].…”

“…CXCL12 was often incorporated into GAG-containing hydrogels [76,78,79] and MMP activity was used for hydrogel degradability [102,[106][107][108]. Most of these studies dealt with non-stabilized CXCL12, few used the [S4V] stabilization in combination with biomaterial applications [35,77].…”