Supplement to:
Myelin Localization of
Peptidylarginine Deiminases 2 and 4: Comparison of PAD2 and PAD4 Activities
WoodDorothy D.1, Ackerley Cameron A.2, van den Brand Ben1, Zhang Li1, Raijmakers Reinout3, Mastronardi Fabrizio G.1 and Moscarello Mario A.1*
1. The Hospital for Sick Children, Molecular Structure and Function, 555 University Avenue, Toronto, Canada
2. Department of Pathology, The Hospital for Sick Children,
555 University Avenue, Toronto, Canada
3. Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, NL-6500 HB Nijmegen, The Netherlands
Running Title: Myelin Localization and Activities of Peptidyarginine Deiminases 2 and 4
Supported by: Canadian Institute of Health Research (CIHR) to MAM and Multiple Sclerosis Society of Canada (MSSC) grant to MAM and FGM
*Correspondence: M.A. Moscarello, Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario Canada M5G 1X8
Email:
Figure 1
A. Immuno electron microscopic control using secondary antibody conjugated with gold particles alone. The arrows indicate non-specific binding of the anti-rabbit-gold antibody. Bar= 500nm
B. Immuno electron microscopic control using anti-PAD2 competed with PAD2 protein. The arrows indicate binding of secondary gold-conjugated antibody.
Bar= 500 nm
Figure 2
Representative immuno slot blots of myelin proteins from normal and MS brains (two individuals each) showing duplicate immunoreactivities to citrulline, PAD2 and PAD4.
Supplementary Table I Sites of citrullination on MBP C-1 using recombinant PAD2 and PAD4 enzymes in aqueous solution.
PAD2 in solution
1 6 11 16 2126 31 36 41 46
asqkr psqrh gskyl atast mdhar hgflp rhrdt gilds igrff ggdrg
51 56 61 66 71 76 81 86 91 96
apkrg sgkds hhpar tahyg slpqk shgrt qdenp vvhff knivt prtpp
101 106 111 116 121 126 131 136 141 146
psqgk gRGLS LSRFS WGaeg qrpgf gyggr asdyk sahkg fkgvd aqgtl
151 156 161 166
skifk lggrd srsgs pmarr
List of modified peptides
SequenceMass(M+H)+Intensity(Counts)
asqkrpsqr1100.68228
asqkrpsqrhgsk1510.76462
rpsqrhgskylatastmdhar2372.16189
rpsqrhgskylatastmdharhgflpr3080.62166
ylatastmdharhgflpr2045.01198
ylatastmdharhgflpr2046.001632
hgflprhr1020.55941
hgflprhrdtgildsigr2049.031269
hrdtgildsigrffggdr2020.01433
hrdtgildsigrffggdr2021.03142
hrdtgildsigrffggdrgapk2373.24148
Dtgildsigrffggdrgapk2080.0491
Dtgildsigrffggdrgapk2081.062540
Dtgildsigrffggdrgapk
(R10-Cit, R16-Methyl)2094.11168
Dtgildsigrffggdrgapk
(R10-Cit, R16-di-Methyl)2108.06107
DtgildsigrffggdrgapkR2237.11142
dtgildsigrffggdr1726.86204
ffggdrgapk1052.521960
dshhpartahygslpqk1902.932529
shgrtqdenpvvhffk1898.921336
nivtprtpppsqgk1492.82367
prtpppsqgk1065.55499
gRGLSLSR (R2-Cit)846.481327
gRGLSLSR (R2-Methyl)859.522364
gRGLSLSR (R2-di-Methyl)873.532342
gRGLSLSRFSWGaegqrpgfgyggr2657.32766
gRGLSLSRFSWGaegqrpgfgyggr2658.29240
gRGLSLSRFSWGaegqrpgfgyggr2671.341433
(R2-Methyl, R8, R17-Cit)
gRGLSLSRFSWGaegqrpgfgyggr2685.38642
(R2-di-Methyl, R8, R17-Cit)
gRGLSLSRFSWGaegqrpgfgyggr2670.351701
(R2-Methyl, R8-Cit)
gRGLSLSRFSWGaegqrpgfgyggr2684.351555 (R2-di-Methyl, R8-Cit)
gRGLSLSRFSWGaegqrpgfgyggr2669.32100
(R2-Methyl)
gRGLSLSRFSWGaegqrpgfgyggr2683.41299
(R2-di-Methyl)
FSWGaegqrpgfgyggrasdyk2394.111091
FSWGaegqrpgfgyggrasdyk2395.112018
lggrdsrsgspmarr1603.9050
lggrdsrsgspmarr1604.81110
lggrdsrsgspmarr1605.79287
lggrdsrsgspmarr1606.781630
dsrsgspmar1064.50113
dsrsgspmarr1220.51113
dsrsgspmarr1221.581251
dsrsgspmarr1222.571177
spmarr718.3616
sgspmarr862.43116
PAD4 in solution
1 6 11 16 2126 31 36 41 46
asqkr psqrh gskyl atast mdhar hgflp rhrdt gilds igrff ggdrg
51 56 61 66 71 76 81 86 91 96
apkrg sgkds hhpar tahyg slpqk shgrt qdenp vvhff knivt prtpp
101 106 111 116 121 126 131 136 141 146
psqgk gRGLS LSRFS WGaeg qrpgf gyggr asdyk sahkg fkgvd aqgtl
151 156 161 166
skifk lggrd srsgs pmarr
List of modified peptides
Sequence Mass(M+H)+ Intensity(Counts)
ylatastmdharhgflpr2045.022216
ylatastmdharhgflprhrdtgildsigr3367.702689
atastmdharhgflpr1768.842111
hgflprhrdtgildsigr2049.062171
HGFLPRHRDTGILDSIGRFFGGDRGAPK3083.65482
HRDTGILDSIGRFFGGDR2020.00138
HRDTGILDSIGRFFGGDRGAPKR2531.10389
DTGILDSIGRFFGGDRGAPK2081.04173
LDSIGRFFGGDRGAPK1694.8671
FFGGDRGAPK1052.522804
DSHHPARTAHYGSLPQK1902.93362
RGSGKDSHHPARTAHYGSLPQK2388.29157
SHGRTQDENPVVHFFK1898.931993
SHGRTQDENPVVHFFKNIVTPR2579.45276
tqdenpvvhffknivtpr2142.161210
nivtprtpppsqgk1492.801759
gRGLSLSR846.48834
gRGLSLSR (Methyl)859.511883 gRGLSLSR (di-Methyl) 873.53 2124 gRGLSLSRFSWGaegqr (R2-methyl, R8-Cit) 1879.02 1386
gRGLSLSRFSWGaegqr (R2, R8-Cit)1866.00381
gRGLSLSRFSWGaegqr (R2-di-Methyl, R8-Cit)1893.041354
GRGLSLSRFSWGAEGQRPGFGYGGR (R2, R8-Cit)2657.382684
GRGLSLSRFSWGAEGQRPGFGYGGR (R2-methyl)2669.441369
GRGLSLSRFSWGAEGQRPGFGYGGR
(R2-methyl, R17-Cit)2670.4641
GRGLSLSRFSWGAEGQRPGFGYGGR
(R2-methyl, R8-Cit)2670.49218
GRGLSLSRFSWGAEGQRPGFGYGGR
(R2-di-methyl, R8-Cit)2684.47350
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2-methyl, R8, R25-Cit)3235.691751
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2, R8, R25-Cit)3222.68513
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2, R8, R17-Cit)3222.73108
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2, R8, R17, R25-Cit)3223.72895
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2-Methyl, R8, R17, R25-Cit)3236.7460
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2-di-methyl, R8, R25-Cit)3249.70961
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2-di-methyl, R17, R25-Cit)3249.76131
GRGLSLSRFSWGAEGQRPGFGYGGRASDYK
(R2-di-Methyl, R8, R17, R25-Cit)3250.72266
SLSRFSWGAEGQRPGFGYGGR (R4-Cit)2273.14140
GLSLSRFSWGAEGQRPGFGYGGR (R6-Cit)2443.3380
SLSRFSWGAEGQRPGFGYGGRASDYK
(R4, R21-Cit)2838.43248
SRFSWGAEGQRPGFGYGGRASDYK
(R2, R19-Cit)2638.30208
GLSLSRFSWGAEGQRPGFGYGGRASDYK
(R6, R23-Cit)3008.56311
FSWGAEGQRPGFGYGGRASDYK
(R17-Cit)2394.072048
FSWGAEGQRPGFGYGGRASDYK
(R9-Cit)2394.2240
PGFGYGGRASDYK
(R8-Cit)1375.68683
lggrdsrsgspmarr1606.78704
LGGRDSRSGSPMARR
(R4,R7,R14,R15-Cit, M12-Ox)1622.8313
SGSPMARR (R7-Cit)862.4626
The starting substrate for the invitro deimination was the component-1 isomer of human MBP. This isomer when isolated from white matter does not contain citrulline. The protein was treated with PAD2 and PAD4 subjected to trypsinization and peptides identified by mass spectrometry.
The underlined red R (R107) in the GRG sequence is the site of deimination and methylation. Extensive treatment with either PAD2 or PAD4 did not remove monomethyl or dimethyl groups from R107.