Development of affinity-based delivery of NGF from a chondroitin sulfate biomaterial.

Chondroitin sulfate is a major component of the extracellular matrix in both the central and peripheral nervous systems. Chondroitin sulfate is upregulated at injury, thus methods to promote neurite extension through chondroitin sulfate-rich matrices and synthetic scaffolds are needed. We describe the use of both chondroitin sulfate and a novel chondroitin sulfate-binding peptide to control the release of nerve growth factor. Interestingly, the novel chondroitin sulfate-binding peptide enhances the controlled release properties of the chondroitin sulfate gels. While introduction of chondroitin sulfate into a scaffold inhibits primary cortical outgrowth, the combination of chondroitin sulfate, chondroitin sulfate-binding peptide and nerve growth factor promotes primary cortical neurite outgrowth in chondroitin sulfate gels.

Chondroitin sulfate-binding peptides block chondroitin 6-sulfate inhibition of cortical neurite growth.

Chondroitin sulfate (CS) expression is increased in the glial scar following spinal cord injury demonstrating the importance understanding the role of CS in the central nervous system (CNS). There have been conflicting studies on the effects of the most abundant types of CS, chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), found in the CNS. In this study, the effects of C4S and C6S on rat embryonic day 18 cortical neurons were investigated. C4S had no effect on neuron behavior whereas C6S inhibited neurite outgrowth at higher concentrations (>10mug/ml). Two C6S-binding peptides (C6S-1 and C6S-2) were tested for their ability to block the inhibitory activity of C6S on neurite outgrowth. Neurons cultured with C6S and C6S-binding peptide at higher peptide concentrations had neurite lengths similar to neurons cultured without C6S. Therefore, the C6S-binding peptides were effective at blocking the inhibitory activity of C6S. The C6S-1 peptide had a higher binding affinity than the C6S-2 peptide and was consequently more effective at blocking C6S inhibition of neurite growth. To date, this is the first study to employ an alternative strategy from enzymatic digestion of CS chains to increase neurite outgrowth. These studies warrant further investigation of the use of C6S-binding peptides to increase nerve regeneration following spinal cord injury.

Identification and sequence composition characterization of chondroitin sulfate-binding peptides through peptide array screening.

Chondroitin sulfate (CS) is an important glycosaminoglycan that has been implicated in several disease processes, such as cancer and spinal cord injury. However, few studies have characterized CS-binding protein and peptide sequences for diagnostic and therapeutic use. In this study, peptide array screening, affinity capillary electrophoresis, and statistical analysis were used to both identify and characterize C6S-binding peptides for sequence composition. The compositional characterization results showed that Phe, Arg, and Tyr all had a significantly high rate of occurrence in the "high binding" affinity peptides, while tryptophan and lysine were significantly underrepresented in this population. Peptides modified with alanine point mutations for Phe, Arg, and Tyr all had lower C6S-binding affinities than the original peptides, demonstrating that these amino acids are all important for C6S binding. Several peptides were designed that substituted Arg for Lys and Phe or Tyr for Trp to create peptides with higher binding affinity. The peptides with the Arg substitution all had improved binding affinities while the Phe/Tyr substitution decreased C6S-binding affinity. Further analysis showed that the increased occurrence of Phe and Tyr in the "high affinity" peptides was dependent upon their positions both within the peptide sequence and in relation to other critical amino acids. Finally, a motif (ABBAA) was suggested for C6S-binding peptides where A represents any aromatic amino acid and B any basic amino acid. The results demonstrate that the methodology developed in this study for sequence composition analysis is an effective technique for the characterization of the interaction between peptides and CS.

A novel assay to probe heparin-peptide interactions using pentapeptide-stabilized gold nanoparticles.

In this article, we present a novel assay to probe the interactions between heparin and heparin-binding peptides based on CALNN pentapeptide-stabilized gold nanoparticles. This assay relies on rapid aggregation of gold nanoparticles and dramatic retardation in the presence of a large excess of heparin due to the binding of peptides to heparin. Using this method, the dissociation constant ( K d) and melting temperature ( T m) of three different peptides against heparin were determined. The results from capillary electrophoresis demonstrated that K d values measured by this method were comparatively accurate. It was found that the peptide with the lowest K d did not have the highest T m. Structural analysis by circular dichroism was performed to explain this phenomenon. A comparison with the results from affinity chromatography indicates that electrostatic interactions only are not the major determinant of the affinity between heparin and peptide, but other interactions such as hydrogen-bonding and hydrophobic interactions may play important roles in the overall interactions. This novel assay is inexpensive, label-free, and easy to implement in the laboratories, does not suffer precipitation of the heparin-peptide complex or their conformational changes caused by surface immobilization, and is expected to be a useful complement to other existing methods.