Resolution of disulfide heterogeneity in Nogo receptor I fusion proteins by molecular engineering

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential t...

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Bibliographic Details
Published in:Biotechnology and applied biochemistry 2010-09, Vol.57 (1), p.31-45
Main Authors: WEINREB, Paul H, DINGYI WEN, RUIZHONG WANG, PORRECA, Frank, SILVIAN, Laura, MEIER, Werner, BLAKE PEPINSKY, R, LEE, Daniel H. S, FANG QIAN, WILDES, Craig P, GARBER, Ellen A, WALUS, Lee, JUNG, Mi-Young, WANG, Joy, RELTON, Jane K, AMATUCCI, Joseph
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Behavior, Animal - drug effects
Biological and medical sciences
Biotechnology
Crystallography, X-Ray
Disease Models, Animal
Disulfides - metabolism
Electrophoresis, Polyacrylamide Gel
Fundamental and applied biological sciences. Psychology
GPI-Linked Proteins - chemistry
GPI-Linked Proteins - genetics
Humans
Male
Methods. Procedures. Technologies
Molecular Sequence Annotation
Molecular Sequence Data
Myelin Proteins - chemistry
Myelin Proteins - genetics
Nogo Receptor 1
Protein engineering
Protein Engineering - methods
Protein Stability
Rats
Rats, Sprague-Dawley
Receptors, Cell Surface - chemistry
Receptors, Cell Surface - genetics
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Recombinant Fusion Proteins - pharmacology
Spinal Cord Injuries - drug therapy
Spinal Nerve Roots - injuries
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Summary:NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys²⁶⁶ and Cys³⁰⁹ with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.
ISSN:0885-4513
1470-8744
DOI:10.1042/BA20100061