maldi-tof mass spectrometry to map disulfide formation in a recombinant human neonatal fc receptor...
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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 Countries2nd ESF Functional Genomics and Disease – satellite meeting
Oslo, 6th September 2005
Jan T Andersen, Inger SandlieInstitute of Molecular Biosciences, UiO
Sune Justesen, Søren Buus Institute of Medical Microbiology and Immunology, University of Copenhagen
Anders Holm, Burkhard FleckensteinInstitute of Immunology, UiO
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
β2-microglobulin is essential for FcRn function
Structure of the heterodimeric FcRn
N
C48 C251C96 C159 C198 C252
C
α1-domain α2-domain α3-domain TM CP
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).
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
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
Soluble wild-type (wt):15 possible SS bonds
Soluble double mutant (mut):6 possible SS bonds
1 possible disulfide bond2m
C48 C251C96 C159 C198 C252
HAT
C48S C251SC96 C159 C198 C252
HAT
C25 C80
HAT
N
C48 C251C96 C159 C198 C252
C
α1-domain α2-domain α3-domain TM CP
wild-type (wt)
Constructs and possible disulfide bonds within the hFcRn heavy chain and h2m
Heterologous expression of soluble hFcRn heavy chain and 2m in E. coli and disulfide assisted oxidative refolding
0 1 2 3 hours
hFcRnheavy chainmutant
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.
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
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
The expected disulfide bond in 2m is proven
No signals corresponding to free cysteine residues were found in 2m.
SNFLNCYVSGFHPSDIEVDLLK DEYACRC25 C80
MH+ 3250.5 SNFLNCYVSGFHPSDIEVDLLK DEYACRC25 C80
SNFLNCYVSGFHPSDIEVDLLK DEYACRC25 C80
MH+ 3250.5
1122.642
1946.963
1674.814
1475.802
3250.504
2497.208
1010.3422203.043
2780.348
844.7632940.311
1854.911
3348.631
1383.725 2318.1192681.198
3052.616
00101-00200\00188bf\1SRef
0
2000
4000
6000
8000
Inte
ns.
[a.u
.]
750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250
m/z
3250.504
0
1000
2000
3000
4000
5000
6000
Inte
ns.
[a.u
.]
3245 3250 3255 3260
m/z
C25 C80
HAT
Both correct disulfide bonds were observed in the mut-hFcRn
1SRef
0
10
20
30
40
50
60
Inte
ns. [a
.u.]
5176.453 2SRef
500
1000
1500
2000
2500
3000
Inte
ns. [a
.u.]
5173.601 3SRef
1000
2000
3000
4000
Inte
ns. [a
.u.]
1SLin Raw
0.2
0.4
0.6
0.8
1.0
4x10
Inte
ns. [a
.u.]
5000 5100 5200 5300 5400
m/z
5176.42
Linear Modeexp. MH+ 5175.82No signals corresponding to wrong
disulfide bonds were detected.
3891.453
1SRef
500
1000
1500
2000
2500
Inte
ns. [a
.u.]
3891.098
2SRef
0
1000
2000
3000
4000
5000
Inte
ns. [a
.u.]
3891.1183SRef
0
1000
2000
3000
4000
Inte
ns. [a
.u.]
3880 3885 3890 3895 3900 3905 3910
m/z
1SRef
0
10
20
30
40
50
60
Intens. [a.u.]
5176.453 2SRef
500
1000
1500
2000
2500
3000
Intens. [a.u.]
5173.601 3SRef
1000
2000
3000
4000
Intens. [a.u.]
1SLin Raw
0.4
0.6
0.8
1.0
4x10
Intens. [a.u.]
5165 5170 5175 5180 5185 5190
m/z
exp. MH+ 3890.95 exp. MH+ 5172.52
ARPSSPGFSVLTCSAFSFYPPELQLR
SGDEHHYSCIVQHAGLAQPLR
C198
C252
C48S C251SC96 C159 C198 C252
HAT
GPYTLQGLLGCELGPDNTSVPTAK
ELTFLLFSCPHR
C96
C159
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 -CH2-CONH2
Detection of cysteine residues participating in disulfide formation
which are alkylated by –CH2-COOH
GPYTLQGLLGCELGPDNTSVPTAKC96
CH2-CONH2
GPYTLQGLLGCELGPDNTSVPTAKC96
CH2-CONH2
GPYTLQGLLGCELGPDNTSVPTAKC96
CH2-COOH
GPYTLQGLLGCELGPDNTSVPTAKC96
CH2-COOH
MH+ 2488.2 MH+ 2489.2m = + 1 Da
How complete is the formation of a disulfide bond?
C96-peptide-CH2-COOH:exp. MH+ 2489.24
C159-peptide-CH2-COOH:exp. MH+ 1520.77
2489.321
0
2000
4000
6000
Inte
ns. [a
.u.]
2489.334 2SRef
0
1000
2000
3000
Inte
ns. [a
.u.]
2470 2475 2480 2485 2490 2495 2500 2505
m/z
2489.33
1519.81
1520.796
0
1
2
3
4x10
Inte
ns. [
a.u.
]
1520.813 2SRef
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
1510.0 1515.0 1520.0 1525.0 1530.0
m/z
1520.81
Formation of the first disulfide bond in mut-hFcRn is not fully complete
free Cys
1. Disulfide bondSS bonded Cys
GPYTLQGLLGCELGPDNTSVPTAK
ELTFLLFSCPHR
C96
C159
C198-peptide-CH2-COOH:theoret. MH+ 2915.46
C252-peptide-CH2-COOH:theoret. MH+ 2376.13
2376.181
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns. [
a.u.
]2376.202 2SRef
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
2360 2365 2370 2375 2380 2385 2390
m/z
2376.20
2915.622
0
2000
4000
6000Inte
ns. [
a.u.
]
2915.630 2SRef
0
1000
2000
3000
Inte
ns. [
a.u.
]
2910 2915 2920 2925 2930 2935
m/z
2915.63
Complete disulfide bond
formation
2. Disulfide bondARPSSPGFSVLTCSAFSFYPPELQLR
SGDEHHYSCIVQHAGLAQPLR
C198
C252
A pronounced and unexpected disulfide bond between the vicinal C251 and C252 was found in the wt-hFcRn
1459.733 1691.811
1871.939
2332.099
2780.301
954.155
1342.776
2166.100
2674.2811249.687
1112.6663891.121
2053.025 3196.5852914.5153574.918
00498ns\0_D7\1\1SRef
0
1
2
3
4
5
4x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500
m/z
α1 α2 α3
C48 C251C96 C159 C198 C252
HAT
The vicinal disulfide bond (C251-C252) was confirmedby MALDI-TOF/TOF-MS
110.178
663.321
2073.108
526.270
962.502
368.264
826.133
175.160754.418301.155
1943.906
1115.485
1806.9411370.548 1507.7232175.996
895.412 1669.835
2315.859
436.169
765.298
1043.880
b68059sa\0_H10\0\2332.1000.LIFT\fast
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [a
.u.]
250 500 750 1000 1250 1500 1750 2000 2250
m/z
y1
y3
y18
MH+
y19
y17
y16y15
y14
y13:missing !
y12y11y10
y9y8
y7
y6y5
110.178
663.321
2073.108
526.270
962.502
368.264
826.133
175.160754.418301.155
1943.906
1115.485
1806.9411370.548 1507.7232175.996
895.412 1669.835
2315.859
436.169
765.298
1043.880
b68059sa\0_H10\0\2332.1000.LIFT\fast
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [a
.u.]
250 500 750 1000 1250 1500 1750 2000 2250
m/z
y1
y3
y18
MH+
y19
y17
y16y15
y14
y13:missing !
y12y11y10
y9y8
y7
y6y5
S G D E H H Y C C I V Q H A G L A Q P L R
y-fragments 1356810 791112141516171819
missing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L R
y-fragments 1356810 791112141516171819
missing
y-fragments 1356810 791112141516171819
missingmissing
S G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missing
y-fragments 1356810 791112141516171819
missingmissing110.178
663.321
2073.108
526.270
962.502
368.264
826.133
175.160754.418301.155
1943.906
1115.485
1806.9411370.548 1507.7232175.996
895.412 1669.835
2315.859
436.169
765.298
1043.880
b68059sa\0_H10\0\2332.1000.LIFT\fast
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [a
.u.]
250 500 750 1000 1250 1500 1750 2000 2250
m/z
y1
y3
y18
MH+
y19
y17
y16y15
y14
y13:missing !
y12y11y10
y9y8
y7
y6y5
110.178
663.321
2073.108
526.270
962.502
368.264
826.133
175.160754.418301.155
1943.906
1115.485
1806.9411370.548 1507.7232175.996
895.412 1669.835
2315.859
436.169
765.298
1043.880
b68059sa\0_H10\0\2332.1000.LIFT\fast
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [a
.u.]
250 500 750 1000 1250 1500 1750 2000 2250
m/z
y1
y3
y18
MH+
y19
y17
y16y15
y14
y13:missing !
y12y11y10
y9y8
y7
y6y5
S G D E H H Y C C I V Q H A G L A Q P L R
y-fragments 1356810 791112141516171819
missing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L R
y-fragments 1356810 791112141516171819
missing
y-fragments 1356810 791112141516171819
missingmissing
S G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missing
y-fragments 1356810 791112141516171819
missingmissing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missing
y-fragments 1356810 791112141516171819
missing
S G D E H H Y C C I V Q H A G L A Q P L RS G D E H H Y C C I V Q H A G L A Q P L Ry-fragments 1356810 791112141516171819
missingmissing
y-fragments 1356810 791112141516171819
missingmissing
Recorded on a Ultraflex MALDI-TOF/TOF-instrument (Bruker) in the laboratory of Peter Roepstorff,
Odense, Denmark
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 C251SC96 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 C251C96 C159 C198 C252
HAT
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
DTTIodomethyl-Fluorophore 2
HAT
digestion
Quantification and identification byHPLC-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:
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
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.
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
Heterologous expression of soluble hFcRn heavy chain and 2m in E. coli and disulfide assisted oxidative refolding
0 1 2 3 hours
hFcRnheavy chainmutant
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 h2m.
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