1. MALDI-TOF mass spectrometry to map disulfide formation in a recombinant human neonatal Fc receptor refolded in vitro Study of protein conformation by MS Functional Genomics in the Nordic Countries 2nd ESF Functional Genomics and Disease – satellite meeting Oslo, 6th September 2005 Jan T Andersen, Inger Sandlie Institute of Molecular Biosciences, UiO Sune Justesen, Søren Buus Institute of Medical Microbiology and Immunology, University of Copenhagen Anders Holm, Burkhard Fleckenstein Institute of Immunology, UiO

2. Crystal Structure and Immunoglobulin G Binding Properties of the Human Major Histocompatibility Complex- Related Fc Receptor, by Anthony P. West, Jr. and Pamela J. Bjorkman (2000) Biochemistry 39, 9698 - 9708  The neonatal Fc receptor (FcRn) is a MHC class I related glycoprotein  Unlike MHC class I, FcRn is nonpolymorphic and lacks a functional peptide binding groove  β 2 -microglobulin is essential for FcRn function Structure of the heterodimeric FcRn N C48 C251 C96 C159 C198 C252 C α 1 -domainα 2 -domainα 3 -domainTMCP

3. Functions of the human neonatal Fc receptor Multiple roles for the major histocompatibility complex class I- related receptor FcRn. by Ghetie V, Ward ES (2000) Annu Rev Immunol 18, 739-66. Review. Immunoglobulin transport across polarized epithelial cells. by Rojas R, Apodaca G (2002) Nat Rev Mol Cell Biol 3, 944-55. Review.  Regulation of the IgG-biodistribution. IgG recycling and/or degradation in endothelial cells.  Binding to the Fc portion of IgG within acidic intracellular compartments (pH 6.0).  Release of IgG upon exposure to the slightly basic extracellular environment (pH 7.4)  Birectional epithelial transport of IgG/IgG-Ag over mucosal surfaces.  Transplacental transport of IgG from mother to fetus (passive immunization).

4. Major objectives of the study  Establish a protocol to generate large amounts of soluble, functional recombinant shFcRn  Characterize the folding of the shFcRn heavy chain by mass spectrometry

5.  The FcRn heavy chain is functional when expressed as a soluble truncated form in eukaryotic cells.  Eukaryotic expression gives low yield at a high cost. Bacteria, however, lack the folding machinery available in eukaryotic cells.  MHC class I has been expressed in E. coli and refolded from inclusion bodies in vitro: Refolding of correctly oxidized MHC class I heavy chains to the native structure in the presence of  2m (Ferre et al., 2003, Protein Sci. 12, 551-9)  Expression, extraction and in vitro refolding are affected by the number of cysteine residues in the recombinant protein. Background

6. Soluble wild-type (wt): 15 possible SS bonds Soluble double mutant (mut): 6 possible SS bonds 1 possible disulfide bond  2m C48 C251 C96 C159 C198 C252 HAT C48S C251S C96 C159 C198 C252 HAT C25 C80 HAT N C48 C251 C96 C159 C198 C252 C α 1 -domainα 2 -domainα 3 -domainTMCP wild-type (wt) Constructs and possible disulfide bonds within the hFcRn heavy chain and h  2m

7. Heterologous expression of soluble hFcRn heavy chain and  2m in E. coli and disulfide assisted oxidative refolding 0 1 2 3 hours hFcRn heavy chain mutant 1) EXPRESSION IN INCLUSION BODIES  IPTG induction  IMAC, HIC and SEC purification  all steps performed under denaturing but non-reducing conditions  gram levels of hFcRn heavy chain (wt and mut)  Purified denatured shFcRn heavy chains were diluted into a solution with an excess of β2m.  hFcRn heavy chains fold up on  2m.  Purification of folded heterodimers by SEC. Non-reducing SDS-PAGE. Eluted fractions from SEC purification of shFcRn (C48S/C251S). Heterodimeric fraction F: 38-54 55-67 74-84 shFcRn (WT): 2.6 mg shFcRn (C48S/C251S): 22.2 mg 2) REFOLDING The correct folding of hFcRn is dependent on the presence of  2m.

8. MALDI-TOF mass spectrometry to map disulfide formation in a recombinant human neonatal Fc receptor refolded in vitro Does in vitro refolding in the presence of  2m select the correctly folded heavy chains? Ultraflex, MALDI-TOF Bruker Daltonics

9. Major questions addressed by MALDI-TOF MS  Presence and completeness of the expected (correct) disulfide bonds?  Presence of wrong disulfide bonds?  Presence of free cysteine residues – expected vs unexpected? Strategy (I)  Separation of refolded  2m - hFcRn heavy chain heterodimers on a denaturing but non-reducing gel  Excision of the Coomassie-stained bands  Alkylation of free cysteine residues with iodoacetamide  in gel digestion with trypsin  Detection of intact disulfide bonded peptides

10. The expected disulfide bond in  2m is proven No signals corresponding to free cysteine residues were found in  2m. C25 C80 HAT

11. Both correct disulfide bonds were observed in the mut-hFcRn 5176.42 Linear Mode exp. MH+ 5175.82 No signals corresponding to wrong disulfide bonds were detected. exp. MH+ 3890.95exp. MH+ 5172.52 ARPSSPGFSVLT C SAFSFYPPELQL R SGDEHHYS C IVQHAGLAQPL R C198 C252 C48S C251S C96 C159 C198 C252 HAT GPYTLQGLLG C ELGPDNTSVPTA K ELTFLLFS C PH R C96 C159

12. Strategy (II)  Alkylation of free cysteine residues with iodoacetamide  Reduction by DTT and alkylation by iodoacetic acid  in gel digestion with trypsin  Detection of free cysteine residues which are alkylated by -CH 2 -CONH 2  Detection of cysteine residues participating in disulfide formation which are alkylated by –CH 2 -COOH MH+ 2488.2MH+ 2489.2  m = + 1 Da How complete is the formation of a disulfide bond?

13. C96-peptide-CH 2 -COOH: exp. MH+ 2489.24 C159-peptide-CH 2 -COOH: exp. MH+ 1520.77 2489.33 1519.81 1520.81 Formation of the first disulfide bond in mut-hFcRn is not fully complete free Cys 1. Disulfide bond SS bonded Cys GPYTLQGLLG C ELGPDNTSVPTA K ELTFLLFS C PH R C96 C159 C198-peptide-CH 2 -COOH: theoret. MH+ 2915.46 C252-peptide-CH 2 -COOH: theoret. MH+ 2376.13 2376.20 2915.63 Complete disulfide bond formation 2. Disulfide bond ARPSSPGFSVLT C SAFSFYPPELQL R SGDEHHYS C IVQHAGLAQPL R C198 C252

14. A pronounced and unexpected disulfide bond between the vicinal C251 and C252 was found in the wt-hFcRn α1α1 α2α2 α3α3 C48 C251 C96 C159 C198 C252 HAT

15. The vicinal disulfide bond (C251-C252) was confirmed by MALDI-TOF/TOF-MS Recorded on a Ultraflex MALDI-TOF/TOF-instrument (Bruker) in the laboratory of Peter Roepstorff, Odense, Denmark

16. Summary  In  2m and mut-hFcRn  -chain, the correct disulfide bonds were demonstrated.  In mut-hFcRn heavy chain the formation of the first disulfide bond is almost but not fully complete. The second disulfide bond formation is complete. Wrong disulfide bonds were not detected. C48S C251S C96 C159 C198 C252 HAT  In wt-hFcRn  -chain, only the first (correct) disulfide bond was observed. Small signals corresponding to wrong disulfide bonds as well as unexpected free cysteine residues were obtained.  A pronounced and wrong disulfide bond formation between the vicinal C251 and C252 was demonstrated by MALDI-TOF/TOF analysis. C48 C251 C96 C159 C198 C252 HAT

17. Outlook  In the future, disulfide formation in soluble hFcRn expressed both in the prokaryotic and eukaryotic system will be analyzed.  nanoLC-offline-MALDI-TOF will be used to increase the “coverage of conformation” (qualitative approach). HAT Iodomethyl-Fluorophore 1 HAT DTT Iodomethyl-Fluorophore 2 HAT digestion Quantification and identification by HPLC-fluorescence detection-MS  More quantitative studies on the disulfide formation in hFcRn will be performed. Use of two fluorescence dyes to differentially label free and disulfide forming cysteines:

19. Characterization of the shFcRn (wt and mut) by Circular Dicroism and Surface Plasmon Resonance IgG1 was immobilized on a CM5 chip shFcRn (C48S/C251S) was injected in different concentrations. Binding of shFcRn (C48S/C251S) to IgG1 at pH6.0  CD spectra show 52.6% and 46.0% (wt and mut, respectively)  -sheet structures, 14.5% α-helical contribution (for wt and mut)  The functionality of the shFcRn was confirmed by SPR (Biacore): - concentration dependent binding to its ligands: human IgG1, human IgG3 and HSA. - pH dependent binding to ligands: binding at pH 6.0 and no binding pH 7.4.  The bacterially expressed and in vitro refolded shFcRn is functional RU time

21. Functions of the human neonatal Fc receptor Multiple roles for the major histocompatibility complex class I- related receptor FcRn. by Ghetie V, Ward ES (2000) Annu Rev Immunol 18, 739-66. Review. Immunoglobulin transport across polarized epithelial cells. by Rojas R, Apodaca G (2002) Nat Rev Mol Cell Biol 3, 944-55. Review.  Maintenance of IgG- and HSA-homeostasis. Modulation of IgG-biodistribution by recycling and/or degradation in endothelial cells.  Birectional epithelial transport of IgG/IgG-Ag over mucosal surfaces.  Transplacental transport of IgG from mother to fetus (passive immunization).  Binding to the Fc portion of IgG within acidic intracellular compartments (pH 6.0).  Release of IgG upon exposure to the slightly basic extracellular environment (pH 7.4)  The interactions are mediated by histidine residues.

22.  The FcRn heavy chain is functional when expressed as a soluble truncated form in eukaryotic cells.  Eukaryotic expression gives low yield at a high cost. Bacteria, however, lack the folding machinery available in eukaryotic cells.  Expression, extraction and in vitro refolding are affected by the number of cysteine residues in the recombinant protein.  MHC class I has been expressed in E. coli and refolded from inclusion bodies in vitro: Ferre et al. (2003) Protein Sci. 12, 551-9: Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding Strategy: disulfide assisted oxidative refolding of the heavy chain in vitro. Refolding of correctly oxidized MHC class I heavy chains to the native structure in the presence of  2m. Background

23. Heterologous expression of soluble hFcRn heavy chain and  2m in E. coli and disulfide assisted oxidative refolding 0 1 2 3 hours hFcRn heavy chain mutant  Transformation of BL21 (DE3) E. coli  Large scale fermentations (2 L)  Induction of expression by IPTG  IMAC, HIC and SEC purification  gram levels of hFcRn heavy chain wt and mut.  The correct refolding to the native structure is dependent on the presence of  2m.  Purified shFcRn heavy chains (denatured but oxidized) were diluted into a solution with an excess amount of hβ2m.  hFcRn heavy chains fold up on h  2m.  Purification of folded heterodimers by SEC. SDS-PAGE. Eluted fractions from SEC purification of shFcRn (C48S/C251S). Heterodimeric fraction F: 38-54 55-67 74-84 shFcRn (WT): 2.6 mg shFcRn (C48S/C251S): 22.2 mg