High binding affinities of GAG toward extracellular regulatory proteins are governed by recognition diversity, sulfation pattern, length, and anomeric functionalization.

The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.

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.

B iallelic pathogenic variants within MAPKBP1, encoding mitogen-activated protein kinase binding protein 1, were recently reported to cause juvenile, late-onset, cilia-independent nephronophthisis ([NPH], Mendelian

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