The rational design of affinity-attenuated OmCI for the purification of complement C5

The Journal of Biological Chemistry
Alex MacphersonAlastair D G Lawson

Abstract

Complement component C5 is the target of the mAb eculizumab and is the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to and potently inactivates C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared with existing methods. However, breaking the high-affinity interaction requires conditions that risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site-directed mutagenesis, and characterized mutants using a range of biophysical techniques, as well as functional assays. Our biophysical analyses suggest that the C5-OmCI interaction is complex with potential for multiple binding modes. We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease or entirely abrogate formation of the complex. The affinity-attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions th...Continue Reading

References

Jan 1, 1975·Scandinavian Journal of Immunology·A G Sjöholm
Feb 14, 1991·Nature·N P Gerard, C Gerard
May 1, 1983·Immunobiology·A Dessauer, U Rother
Nov 5, 1999·The Journal of Biological Chemistry·R G DiScipioN K Rushmere
Nov 4, 2000·Biochimica Et Biophysica Acta·D R FlowerC E Sansom
May 23, 2001·International Immunopharmacology·N Rawal, M K Pangburn
Nov 21, 2002·Biochemical Society Transactions·M K Pangburn, N Rawal
Jul 25, 2003·The Journal of Biological Chemistry·Nenoo Rawal, Michael K Pangburn
Jun 14, 2005·Journal of Molecular Recognition : JMR·Annica Onell, Karl Andersson
Jan 12, 2007·The Journal of Biological Chemistry·Natalie J HepburnClaire L Harris
Apr 21, 2007·Journal of Molecular Biology·Pietro RoversiSusan M Lea
Jun 10, 2008·Nature Immunology·Folmer FredslundGregers R Andersen
Oct 12, 2010·Canadian Journal of Ophthalmology. Journal Canadien D'ophtalmologie·David LockingtonHugh Willison
Jan 11, 2011·The EMBO Journal·Nick S LaursenGregers R Andersen
May 10, 2011·Molecular Immunology·Trent M WoodruffFrancesco Tedesco
Jul 1, 2007·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Urmi Roy, Linda A Luck
Apr 14, 2012·The Journal of Biological Chemistry·Alexander E AleshinRobert C Liddington
Apr 30, 2013·The Journal of Biological Chemistry·Pietro RoversiMiles A Nunn
Nov 13, 2013·Methods in Molecular Biology·Lars Sottrup-Jensen, Gregers Rom Andersen
Feb 14, 2014·The New England Journal of Medicine·Jun-ichi NishimuraYuzuru Kanakura
Oct 24, 2015·Nature Reviews. Drug Discovery·B Paul Morgan, Claire L Harris
Nov 5, 2015·Current Opinion in Organ Transplantation·Christopher K Johnson, Nicolae Leca
Feb 5, 2016·Nature Communications·Marina SernaDoryen Bubeck
Mar 29, 2016·Nature Structural & Molecular Biology·Matthijs M JoreSusan M Lea
May 20, 2016·The Journal of Immunology : Official Journal of the American Association of Immunologists·Janus Asbjørn Schatz-JakobsenGregers Rom Andersen
Jun 21, 2016·Seminars in Immunology·Daniel Ricklin, John D Lambris
Aug 12, 2016·Scientific Reports·Anna-Luisa VolkJohan Rockberg

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Citations

Sep 5, 2020·PLoS Biology·Alex MacphersonAlastair D G Lawson
Feb 12, 2020·Journal of Chromatography. a·Manuel J B MatosA C A Roque
Feb 12, 2021·ELife·Alex MacphersonJean Mh van den Elsen
Aug 19, 2021·ACS Chemical Biology·Alex MacphersonAlastair D G Lawson

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Methods Mentioned

BETA
ion-exchange
gel-filtration
affinity purification
chip
affinity-purification
differential scanning calorimetry
affinity
gel filtration
Transfection

Software Mentioned

Prism
Molecular Operating Environment ( MOE )
MOE
Biacore
Origin

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