Polysialic acid (PSA) is a unique post-translational modification found on the neural cell adhesion molecule (NCAM) and plays an important physiological role in nervous system development through modulation of neuronal interactions. As a large polyanion, PSA regulates cell-cell and cell-matrix interactions through electrostatic repulsion and steric hindrance. At the same time, a number of proteins are known to act as specific ligands to PSA, including polysialyltransferases, brain-derived neurotrophic factor, and Siglec-11 – a sialic-acid specific lectin that recognizes α-2,8-linked N-acetylneuraminic acid. Efficient small-molecule ligands for selective recognition of PSA have not been developed, possibly due to the relatively high entropic penalty associated with binding. Such ligands or biomolecules, including peptides, may serve as biosensors, modulators in infections caused by PSA-expressing microbes, or novel affinity agents for neural stem cell purification. In this work, we demonstrate the development of PSA-binding peptides through rational design, using epitope mapping of Siglec-11 for understanding of PSA-lectin interaction regions and identification of lead candidates. A library of 15-mer peptides, derived from linear epitope mapping of Siglec-11, was synthesized and screened in high-throughput, wherein screening in a spatially addressable manner on peptide microarrays enabled study of peptide-PSA binding interactions. Lead peptides thus isolated appear to show a degree of overlap with homologous regions of other Siglecs that directly interact with sialic acids. Rational screening leads were further studied using surface plasmon resonance (SPR) spectroscopy. Additionally, phage display screening was used as a complementary random screening approach to determine the similarity of consensus motifs to these leads. PSA-binding peptides isolated through combined random and rational screening approaches were tuned for improved affinity and selectivity through an iterative approach. This encompassed rounds of library design and screening with incorporation of point mutations and boronic acid as a non-natural side chain. The potential of lead peptides to serve as selective affinity agents for purification of neural stem cells, which differ from differentiated cells in PSA modification of NCAM, provides an alternative to antibodies and has implications for small-molecule targeting of specific glycosylation patterns in disease-related applications.

Rational design and development of polysialic acid-binding peptides: Implications in neural stem cell purification

251st National Meeting (2016)

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