Identification and characterization of synthetic chondroitin-4-sulfate binding peptides in neuronal functions

Gabriele Loers,Chengliang Hu,Weijiang Zhao,Weikang Xue,Yonghong Liao,Huifan Shen,Melitta Schachner

doi:10.1038/s41598-018-37685-2

Abstract

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the glial scar after mammalian spinal cord injury, have been suggested to be key inhibitory molecules for functional recovery by impeding axonal regrowth/sprouting and synaptic rearrangements. CSPG-mediated inhibition is mainly associated with the glycosaminoglycan chains of CSPGs, and chondroitin-4-sulfate (C4S) is the predominant sulfated structure that regulates axonal guidance and growth in the adult nervous system. With the aim to find molecules that neutralize the inhibitory functions of C4S, we screened a phage display library for peptides binding to C4S. From the phage clones binding to C4S we selected three peptides for further analysis. We observed that these peptides bind to C4S, but not chondroitin-6-sulfate, heparin sulfate or dermatan sulfate, in a concentration-dependent and saturable manner, whereas the scrambled peptides showed highly reduced or no binding to C4S. The C4S-binding peptides, but not their scrambled counterparts, when added to cultures of mouse cerebellar neurons and human neuroblastoma cells, neutralized the inhibitory functions of the C4S- and CSPG-coated substrate on cell adhesion, neuronal migration and neurite outgrowth. These results indicate that the C4S-binding peptides neutralize several inhibitory functions of CSPGs, suggesting that they may be beneficial in repairing mammalian nervous system injuries.

Highlights

Mammals exhibit poor recovery after injury to the spinal cord due to the presence of growth inhibitors and diminished intrinsic regenerative capacity of mature neurons in the adult central nervous system[1,2,3]
The eluted 300 phage clones were subjected to a further ELISA and 37 clones showing the highest binding to C4S (Fig. 1) were picked and sequenced to determine the sequence of the peptides that they are carrying on their coat protein and that mediate the binding to C4S
Chondroitin sulfate proteoglycans (CSPGs) are considered as major components of inhibitory molecules that restrict neurite growth and neuritogenesis

Summary

Introduction

Mammals exhibit poor recovery after injury to the spinal cord due to the presence of growth inhibitors and diminished intrinsic regenerative capacity of mature neurons in the adult central nervous system[1,2,3]. It is well accepted that glia-derived chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix within the inhibitory glial scar[8] and that inhibition is mainly associated with CSPG’s glycosaminoglycan chains. Chondroitin sulfate (CS) represents the predominant inhibitory glycosaminoglycan (GAG) structure that is expressed at and around central nervous system injury sites. Among the methods that have shown promise in identifying ligands for functionally important molecules is the phage display technology, first introduced by George Smith[17]. This method represents a powerful and unbiased approach to identify peptide ligands for almost any target. Screening of phage display libraries benefits the most varied fields of research, such as peptide drug discovery[21], isolation of high-affinity antibodies[22], identification of biomarkers[23], and vaccine development[24]

Methods

Results

Conclusion