Characterization of a Glycoprotein of TTP4000 – A Soluble Decoy RAGE Antagonist
Abstract only RAGE is a member of the Ig supergene family that binds amyloid beta with nanomolar affinity. Amyloid beta is implicated in the pathology of neurodegenerative Alzheimer's disease. We have developed TTP4000, a novel fusion protein that has similar ligand binding characteristics as s...
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| Published in: | The FASEB journal Vol. 27; no. S1 |
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| Main Authors: | , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
01-04-2013
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| Online Access: | Get full text |
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| Summary: | Abstract only RAGE is a member of the Ig supergene family that binds amyloid beta with nanomolar affinity. Amyloid beta is implicated in the pathology of neurodegenerative Alzheimer's disease. We have developed TTP4000, a novel fusion protein that has similar ligand binding characteristics as sRAGE but a superior pharmacokinetic profile. TTP4000 was shown to significantly reduce soluble, brain Aβ levels in a mouse model of Alzheimers disease which correlates with a decrease in plaque load, reduction in inflammatory cytokine levels and improved measures of behavior. In this study we investigated the role of glycosylation on TTP4000 ligand binding activity. Incubation of wildtype TTP4000 with PNGase F and endoglycosidases to remove glycans results in improved ligand binding activity. Treatment of cells with 2‐deoxy‐D‐glucose results in reduced glycosylation and a slight increase in Aβ binding activity. We also show a relationship between reduction of TTP4000 glycosylation and increased ligand binding activity following mutation of N‐linked glycan sites that prevent glycosylation of TTP4000 at asparagines 2, 58, and 288. The N2Q‐R198A mutant showed 48 fold greater affinity in the Aβ binding assay when compared to wildtype. We also observed reduced expression levels of TTP4000 mutants when too much glycan was removed. The data herein demonstrate the importance of glycosylation for both stability and ligand binding activity in the design of a RAGE peptide antagonist. |
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| ISSN: | 0892-6638 1530-6860 |
| DOI: | 10.1096/fasebj.27.1_supplement.803.2 |