DS-affinity fractionation enriches certain HEp-2 proteins
Proteins were extracted from freshly cultured HEp-2 cells and fractionated with DS-affinity resins. Since DS molecules are poly-anionic, ionic interactions are expected to be a main contributor to DS affinity. We therefore developed an NaCl salt step-gradient method to sequentially dissociate and elute proteins with from DS resins. The identities of proteins in each DS-affinity enriched fraction were obtained by mass spectrometry sequencing.
Proteins with high DS-affinity
From fractions eluted with 1.0 M NaCl from DS-affinity resins, only 7 proteins were identified by mass spectrometry sequencing (Table 1). These include two histone proteins (H4 and H2BE), Scl-70, and Ro/SS-A, all of which are among the most classical nuclear autoantigens (Table 1). Three isoforms of ribosomal proteins are also identified, including 60S ribosomal proteins (L6 and L7) and 40S ribosomal protein S9. Ribosomal proteins are also a class of well-known autoantigens, and heterogenous forms have been reported to be recognize autoantibodies, however, it is not clear exactly which and how many isoforms are autoantigens. L6 and L7 have been reported as autoantigens, whereas S9 awaits further confirmation (Table 1).
Table 1. Proteins identified from DS-affinity enrichment of HEp-2 cell extract
Pept. #*
|
Gene
|
Protein
|
H
|
M
|
L
|
pI
|
Ref.
|
|
|
Nuclear / Chromosome / Mitotic Cell Cycle
|
|
|
|
|
|
10
|
HIST1H4A
|
Histone H4
|
+
|
+
|
|
11.36
|
[18]
|
3
|
HIST2H2BE
|
Histone H2B type 2-E
|
+
|
+
|
|
10.31
|
[19]
|
4
|
H1-2
|
Histone H1.2 (H1F2, HIST1H1C)
|
|
+
|
|
10.94
|
[20]
|
3
|
H2AFY
|
Core histone macro-H2A.1
|
|
+
|
|
9.80
|
[21]
|
2
|
H1-5
|
Histone H1.5 (H1F5, HIST1H1B, DNMT3A)
|
|
+
|
|
10.91
|
[20]
|
2
|
H1F0
|
Histone H1.0
|
|
+
|
|
10.84
|
[22]
|
2
|
H2AFV
|
Histone H2A.V
|
|
+
|
|
10.58
|
[23]
|
2
|
HIST2H3A
|
Histone H3.2
|
|
+
|
|
11.27
|
[24]
|
Ab
|
TOP1
|
DNA topoisomerase I (Scl-70 autoantigen)
|
+
|
|
|
|
[2, 25]
|
5
|
SET
|
Protein SET (Inhibitor of granzyme A-activated DNase, HLA-DR-associated protein II)
|
|
+
|
|
4.22
|
?
|
Ab
|
TRIM21
|
E3 ubiquitin-protein ligase TRIM21 (Ro/SS-A autoantigen)
|
+
|
|
|
|
[2, 26]
|
4
|
SSB
|
Lupus La protein
|
|
+
|
+
|
6.68
|
[27]
|
4
|
ANP32B
|
Acidic leucine-rich nuclear phosphoprotein 32 family member B (putative HLA-DR-associated protein I-2)
|
|
+
|
|
3.93
|
?
|
2
|
PRMT1
|
Protein arginine N-methyltransferase 1 (Histone-arginine N-methyltransferase PRMT1)
|
|
|
+
|
5.18
|
?
|
28
|
DDB1
|
Damage-specific DNA-binding protein 1
|
|
|
+
|
5.14
|
[2]
|
2
|
NPM1
|
Nucleophosmin
|
|
+
|
|
4.64
|
[28]
|
4
|
KPNB1
|
Importin subunit beta-1 (pore targeting complex 97 kDa subunit)
|
|
|
+
|
4.68
|
[29]
|
3
|
PTMA
|
Prothymosin alpha
|
|
|
+
|
3.66
|
[30]
|
3
|
HDGF
|
Hepatoma-derived growth factor (High mobility group protein 1-like 2)
|
|
|
+
|
4.70
|
[31]
|
12
|
ENO1
|
Alpha-enolase
|
|
|
+
|
7.01
|
[2, 32]
|
3
|
MYBBP1A
|
Myb-binding protein 1A
|
|
+
|
|
9.34
|
?
|
4
|
YBX3
|
Y-box-binding protein 3 (Single-strand DNA-binding protein)
|
|
+
|
|
9.77
|
[2, 33]
|
10
|
YWHAE
|
14-3-3 protein epsilon
|
|
|
+
|
4.63
|
[34]
|
5
|
YWHAZ
|
14-3-3 protein zeta/delta
|
|
|
+
|
4.73
|
[35]
|
4
|
YWHAG
|
14-3-3 protein gamma
|
|
|
+
|
4.80
|
[34]
|
3
|
YWHAQ
|
14-3-3 protein theta
|
|
|
+
|
4.68
|
[36]
|
2
|
YWHAB
|
14-3-3 protein beta/alpha (Protein kinase C inhibitor protein 1)
|
|
|
+
|
4.76
|
?
|
4
|
SFN
|
14-3-3 protein sigma (Stratifin)
|
|
|
+
|
4.68
|
?
|
Ab
|
PCNA
|
Proliferating cell nuclear antigen
|
|
+
|
|
|
[2, 37]
|
21
|
XRCC6
|
Ku70 (X-ray repair cross-complementing protein 6, ATP-dependent DNA helicase 2 subunit 1)
|
|
+
|
+
|
6.23
|
[38]
|
10
|
XRCC5
|
Ku80 (Ku86, X-ray repair cross-complementing protein 5, ATP-dependent DNA helicase 2 subunit 2)
|
|
+
|
+
|
5.55
|
[39]
|
4
|
PRKDC
|
DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
|
|
+
|
|
6.75
|
[40]
|
2
|
MCM6
|
DNA replication licensing factor MCM6
|
|
|
+
|
5.28
|
?
|
2
|
MCM2
|
DNA replication licensing factor MCM2
|
|
|
+
|
5.34
|
?
|
2
|
MCM3
|
DNA replication licensing factor MCM3
|
|
|
+
|
5.53
|
?
|
|
|
|
|
|
|
|
|
|
|
RNA Metabolism / Ribonucleoprotein Granule
|
|
|
|
|
|
3
|
RPL6
|
60S ribosomal protein L6
|
+
|
|
|
10.59
|
[41]
|
2
|
RPS9
|
40S ribosomal protein S9
|
+
|
|
|
10.66
|
[42]
|
2
|
RPL7
|
60S ribosomal protein L7
|
+
|
|
|
10.66
|
[43]
|
16
|
RPL5
|
60S ribosomal protein L5
|
|
+
|
|
9.73
|
[44]
|
2
|
MRPL39
|
39S ribosomal protein L39, mitochondrial
|
|
+
|
|
7.56
|
?
|
16
|
PABP4
|
Poly(A)-binding protein 4
|
|
+
|
|
9.31
|
[2]
|
9
|
PABP3
|
Poly(A)-binding protein 3
|
|
+
|
|
9.68
|
[2]
|
12
|
NCL
|
Nucleolin
|
|
+
|
+
|
4.60
|
[45]
|
3
|
ANP32A
|
Acidic leucine-rich nuclear phosphoprotein 32 family member A (putative HLA-DR-associated protein I)
|
|
+
|
|
3.98
|
?
|
3
|
DHX9
|
ATP-dependent RNA helicase A (DEAH box protein 9)
|
|
+
|
|
6.41
|
[46]
|
4
|
HNRNPCL1
|
Heterogeneous nuclear ribonucleoprotein C-like 1
|
|
+
|
|
4.93
|
?
|
3
|
HNRNPU
|
Heterogeneous nuclear ribonucleoprotein U (Scaffold-attachment factor A)
|
|
+
|
+
|
5.76
|
?
|
2
|
HNRNPA3
|
Heterogeneous nuclear ribonucleoprotein A3
|
|
+
|
|
9.10
|
[47]
|
4
|
SYNCRIP
|
Heterogeneous nuclear ribonucleoprotein Q (Synaptotagmin binding cytoplasmic RNA interacting protein)
|
|
|
+
|
8.68
|
?
|
3
|
HNRNPA1
|
Heterogeneous nuclear ribonucleoprotein A1
|
|
|
+
|
9.17
|
[48]
|
2
|
HNRNPR
|
Heterogeneous nuclear ribonucleoprotein R
|
|
|
+
|
8.23
|
[49]
|
2
|
HNRNPA2B1
|
Heterogeneous nuclear ribonucleoprotein A2/B1
|
|
|
+
|
8.97
|
[50]
|
3
|
C1QBP
|
Complement component 1 Q subcomponent-binding protein, mitochondrial (Hyaluronan-binding protein) (Mitochondrial)
|
|
|
+
|
4.74
|
[51]
|
2
|
SUB1
|
Activated RNA polymerase II transcriptional coactivator p15 (PC4, RPO2TC1)
|
|
|
+
|
9.60
|
?
|
12
|
PRPF8
|
Pre-mRNA-processing-splicing factor 8 (U5 snRNP-specific protein 220 kDa)
|
|
|
+
|
8.95
|
[2]
|
2
|
EFTUD2
|
116 kDa U5 small nuclear ribonucleoprotein component
|
|
|
+
|
4.84
|
[52]
|
11
|
SF3B3
|
Splicing factor 3B subunit 3
|
|
|
+
|
5.13
|
[2, 53]
|
3
|
SFRS7
|
Serine/arginine-rich splicing factor 7
|
|
|
+
|
11.83
|
[2, 54]
|
3
|
VIM
|
Vimentin (Cytoskeleton)
|
|
|
+
|
5.05
|
[55]
|
4
|
IQGAP1
|
Ras GTPase-activating-like protein IQGAP1 (Membrane)
|
|
|
+
|
6.08
|
[56]
|
|
|
|
|
|
|
|
|
|
|
Vesicle / ER / Mitochondrion
|
|
|
|
|
|
51
|
CLTC
|
Clathrin heavy chain 1
|
|
|
+
|
5.57
|
[2]
|
2
|
CLTCL1
|
Clathrin heavy chain 2
|
|
|
+
|
4.29
|
?
|
15
|
HSPA8
|
Heat shock cognate 71 kDa protein (LPS-associated protein 1, HSP7C) (ER)
|
|
+
|
|
5.37
|
[57]
|
34
|
HSPA5
|
Endoplasmic reticulum chaperone BiP (Grp-78, Immunoglobulin heavy chain-binding protein)
|
|
|
+
|
5.07
|
[58]
|
25
|
HSPA9
|
Stress-70 protein, mitochondrial (GRP-75, Mortalin)
|
|
|
+
|
5.87
|
[2, 57]
|
9
|
HSP90AA1
|
Heat shock protein HSP 90-alpha (Hsp86, NY-REN-38, LPS-associated protein 2) (Membrane)
|
|
|
+
|
4.94
|
[59]
|
7
|
HSP90AB1
|
Heat shock protein HSP 90-beta (Hsp84, HSP90B, HSPC2) (Membrane)
|
|
|
+
|
4.96
|
[60]
|
5
|
HSP90B1
|
Endoplasmin (GRP-94, Tumor rejection antigen 1)
|
|
|
+
|
4.76
|
[61]
|
4
|
HSP90AA2P
|
Heat shock protein HSP 90-alpha A2 (HSP90AA2, HSPCAL3)
|
|
|
+
|
4.57
|
[62]
|
19
|
CALR
|
Calreticulin (Calregulin, ER resident protein 60)
|
|
|
+
|
6.19
|
[63]
|
12
|
P4HB
|
Protein disulfide-isomerase (cellular thyroid hormone-binding protein)
|
|
|
+
|
4.76
|
[64]
|
6
|
PDIA6
|
Protein disulfide-isomerase A6 (ER protein 5)
|
|
|
+
|
4.95
|
?
|
6
|
PDIA4
|
Protein disulfide-isomerase A4
|
|
|
+
|
4.96
|
?
|
10
|
VCP
|
Transitional endoplasmic reticulum ATPase (Valosin-containing protein)
|
|
|
+
|
5.14
|
[65]
|
5
|
CALM1
|
Calmodulin-1
|
|
|
+
|
4.09
|
[66]
|
3
|
LRPPRC
|
Leucine-rich PPR motif-containing protein, mitochondrial (GP130)
|
|
|
+
|
5.81
|
[67]
|
3
|
CANX
|
Calnexin (Major MHC class I antigen-binding protein p88)
|
|
|
+
|
4.46
|
[68]
|
2
|
ERO1A
|
ERO1-like protein alpha (ER oxidoreductase alpha, disulfide bond formation in ER)
|
|
|
+
|
5.48
|
?
|
2
|
AHSG
|
Alpha-2-HS-glycoprotein (FETUA, fetuin-A)
|
|
|
+
|
5.43
|
[2, 69]
|
2
|
COPG1
|
Coatomer subunit gamma-1 (Golgi)
|
|
|
+
|
5.32
|
?
|
2
|
COPB2
|
Coatomer subunit beta
|
|
|
+
|
5.14
|
[70]
|
3
|
CAPN2
|
Calpain-2 catalytic subunit (Calcium-activated neutral proteinase 2) (Membrane)
|
|
|
+
|
4.87
|
?
|
2
|
PRKCSH
|
Glucosidase 2 subunit beta (Protein kinase C substrate 60.1 kDa protein heavy chain)
|
|
+
|
+
|
4.33
|
?
|
2
|
PPM1G
|
Protein phosphatase 1G (PPM1C)
|
|
|
+
|
4.27
|
[71]
|
15
|
UBA1
|
Ubiquitin-like modifier-activating enzyme 1 (Mitochondrion)
|
|
|
+
|
5.49
|
[2, 72]
|
2
|
PSMA7
|
Proteasome subunit alpha type-7
|
|
|
+
|
8.60
|
[73]
|
2
|
PSMA2
|
Proteasome subunit alpha type-2
|
|
|
+
|
6.91
|
?
|
2
|
PSMA5
|
Proteasome subunit alpha type-5
|
|
|
+
|
4.74
|
?
|
3
|
PSMD6
|
26S proteasome non-ATPase regulatory subunit 6
|
|
|
+
|
5.45
|
?
|
2
|
PSMD13
|
26S proteasome non-ATPase subunit 13
|
|
|
+
|
5.53
|
[74]
|
|
|
|
|
|
|
|
|
|
|
Cytoskeleton
|
|
|
|
|
|
21
|
FLNA
|
Filamin-A (Actin-binding protein 280)
|
|
+
|
+
|
5.70
|
[2, 75]
|
17
|
FLNB
|
Filamin-B (Thyroid autoantigen)
|
|
+
|
|
5.47
|
[2]
|
3
|
LMNA
|
Lamin-A/C (Renal carcinoma antigen NY-REN-32)
|
|
+
|
+
|
6.57
|
[76]
|
28
|
ACTN1
|
Alpha-actinin-1 (Membrane)
|
|
|
+
|
|
[77]
|
22
|
ACTN4
|
Alpha-actinin-4 (Nucleus)
|
|
|
+
|
|
[2, 78]
|
2
|
ACTA2
|
Actin, aortic smooth muscle (alpha-actin 2, cell growth-inhibiting gene 46 protein)
|
|
|
+
|
5.24
|
?
|
2
|
ACTB
|
Actin, cytoplasmic 1 (Beta-actin) (Nucleus)
|
|
|
+
|
5.29
|
[79]
|
2
|
ACTBL2
|
Beta-actin-like protein 2 (Kappa-actin)
|
|
|
+
|
5.39
|
?
|
17
|
SPTAN1
|
Spectrin alpha chain non-erythrocytic 1, fodrin alpha chain
|
|
|
+
|
5.22
|
[80]
|
7
|
MYH9
|
Myosin-9
|
|
|
+
|
5.50
|
[81]
|
4
|
TPM4
|
Tropomyosin alpha-4 chain
|
|
|
+
|
4.67
|
[82]
|
3
|
TPM1
|
Tropomyosin alpha-1 chain
|
|
|
+
|
4.69
|
[83]
|
2
|
TPM3
|
Tropomyosin alpha-3 chain
|
|
|
+
|
4.68
|
[84]
|
2
|
TPM2
|
Tropomyosin beta chain
|
|
|
+
|
4.66
|
?
|
3
|
BASP1
|
Brain acid soluble protein 1 (Neuronal axonal membrane protein NAP-22)
|
|
|
+
|
4.62
|
?
|
3
|
TUBA1C
|
Tubulin alpha-1C chain (alpha-tublin 6)
|
|
|
+
|
4.96
|
[85]
|
2
|
TUBB4B
|
Tubulin beta-2C chain
|
|
|
+
|
4.79
|
[86]
|
*Columns left to right: Number of peptides detected by mass spectrometry sequencing (Ab, confirmed by autoantibody staining [2]); Gene name; Protein name; H (high affinity): eluted with 1.0 M salt; M (medium affinity): eluted with 0.6 M salt; L (low affinity): eluted with 0.4 M salt; Predicted isoelectric point (pI); Literature reference reporting autoantibodies against the protein (“?”: no literature evidence found)
Proteins with medium DS-affinity
From fractions eluted with 0.6 M NaCl from DS-affinity resins, 31 proteins were identified by mass spectrometry (Table 1). Histone H4 and H2B were redundantly identified in both 1.0 M and 0.6 M fractions and thus not counted again. Among these, there are 6 histone autoantigens (H1.0, H1.2, H1.5, H2A.V, H2A.1, and H3.2). Other known protein autoantigens include L5, hnRNP A3, nucleolin, nucleophosmin, lamin A/C, DHX9, PABP4, PABP3, YBX3, DNA-PKcs, PCNA, Ku80 and Ku70, lupus La antigen, filamin A and B, and HSPA8 (Table 1). Several proteins have not been found in literature as reported autoantigens, including SET, PRKCSH, ANP32A, ANP32B, L39, MYBBP1A, and hnRNP protein U and C-like-1.
Proteins with low DS-affinity
From fractions eluted with 0.4 M NaCl from DS-affinity resins, 69 proteins were identified (Table 1). The 8 proteins that were also identified in the 0.6 M fraction are not counted here. These proteins ranged from nuclear, cytosol, mitochondrial, cytoskeleton, to proteasome. Proteins related to cellular cytoskeleton include tubulin, tropomyosin, actin, alpha-actinin 1 and 4, spectrin, vimentin, calmodulin, calreticulin, and myosin, most of which have been previously reported as autoantigens. Several interesting groups of proteins are identified, including DNA replication 3 licensing factor proteins (MCM6, MCM2, and MCM3), 6 14-3-3 proteins (epsilon, zeta/delta, gamma, theta, beta/alpha, and sigma), 6 heat shock proteins (HSPA5, HSPA9, HSP90AA1, HSP90AA2, HSP90AB1, and HSP90B1), and 5 proteasomal proteins (PSMA2, PSMA5, PSMA7, PSMD6, and PSMD13). Several enzymes that function in the ER were identified, including P4HB, PDIA4, PDIA6, ERO1A, AHSG, and VCP. Other interesting proteins include Golgi proteins (COPG1 and COPB2), mitochondrial LRPPRC, growth factor HDGF, C1QBP, IQGAP1, BASP1, and clathrin.
DS affinity strongly enriches for autoantigenic proteins
Overall, this study identified a total of 107 proteins from DS-affinity enrichment of HEp-2 cellular extracts (Table 1). Based on current literature, 78 of 107 (72.9%) proteins are confirmed autoantigens. The rest (29 proteins) are potential autoantigens yet to be verified in future studies.
The DS-affinity HEp-2 proteome shows protein network and functional enrichment
To understand the set of DS-affinity-associated proteins identified in this study, we performed various protein network analyses. From protein association network STRING analysis, 106 nodes (proteins, with HSP90AA2 not recognized) gave rise to 330 edges (protein-protein associations) vs. the expected 142 edges (with average node degree of 6.23, and average local clustering coefficient of 0.531, and PPI enrichment p-value of <1.0e-16). The STRING analysis results indicate that the set of proteins identified from this study have significantly more interactions among themselves than what would be expected of a random set of proteins of similar size drawn from the genome. This insight suggests that DS-affinity proteins may share certain biological functions. While not definitive, it may also indicate that these proteins share functional or biochemical properties (i.e., forming macromolecular charge affinity complexes) and perhaps immunological properties (we have previously shown that DS-autoAg complexes stimulate autoreactive B cells and autoantibody production [1-4]).
The proteins identified from this study are not randomly distributed, but rather can be classified into 4 clusters, chromosome-binding, RNA-binding, vesicle, and cytoskeleton (Fig. 1A). Based on GO Molecular Function and Cellular Component analysis, nuclear proteins are the most significant group. Of the 107, 82 proteins can be found in the nucleus, including 36 DNA-binding, 7 histone-binding, and 28 RNA-binding proteins. In particular, 17 proteins are expressed in the M phase of cell cycle (Fig. 1B). According to Reactome Pathways, 24 proteins can be involved in cell cycle, with 20 potentially attributable to the mitotic phase and 17 protein attributable to the G2/M check points. In addition to nuclear proteins, another prominent group is related to vesicles/granulates. This group comprises of 48 proteins, including 35 vesicle components, 20 vesicle-mediated transporters, and 12 ribonucleoprotein granule proteins (Fig. 1C). Another prominent group are proteins associated with cytoskeleton organization (32/107).
To further capture the relationships among these proteins, Metascape pathway and process enrichment analyses were conducted. The three most prominent ontology clusters identified are mRNA metabolic process regulation, apoptosis, and DNA conformation change (Fig. 2). Other significant clusters include translocation of SLC2A4 to plasma membrane, protein processing in the ER, Nop56p-associated pre-rRNA complex, nucleocytoplasmic transport, Emerin complex 52, C complex spliceosome, DGCR8 multiprotein complex, H2AX complex, telomere maintenance, ACTB-ANP32A-C1QBP-PSMA1-PTMA-PSMA1 complex, DHX9-ADAR-vigilin-DNA-PK-Ku antigen complex, and systemic lupus erythematosus (Fig. 2).
The MCODE (molecular complex detection) algorithm identified four significant networks (Fig. 2). The first MCODE network consists of 23 proteins (CLTC, YWHAB, HSPA9, PABPC3, KPNB1, ACTB, P4HB, ACTA2, UBA1, VCP, H2BC21, COPB2, CLTCL1, YWHAE, PDIA4, PABPC4, YWHAQ, TUBB4B, ENO1, HSPA5, HSPA8, HNRNPA1, and HSP90AA1). This network is likely associated with cellular response to heat stress, vesicle-mediated transport, or kinase maturation complex 1. The second MCODE network consists of 18 proteins (PCNA, MYH9, TPM2, ACTN4, TPM4, IQGAP1, SPTAN1, HSP90B1, XRCC6, PRKDC, PRPF8, DHX9, PSMD13, RPL5, RPS9, COPG1, RPL7, and NPM1). This network is likely associated with PID BARD1 pathway, DHX9-ADAR-vigilin-DNA-PK-Ku antigen complex, or Nop56p-associated pre-rRNA complex. The third MCODE network consists of 12 proteins (CALR, HSP90AB1, ACTN1, YWHAZ, HNRNPA2B1, SRSF7, HNRNPA3, EFTUD2, YWHAG, MCM3, MCM2, and HNRNPR), which are likely associated with mRNA splicing or C complex spliceosome. The fourth MCODE network also consists of 12 proteins (VIM, TPM3, PRMT1, TPM1, H1-2, HNRNPU, SF3B3, TOP1, XRCC5, FLNA, RPL6, and NCL), which are likely associated with Nop56p-associated pre-rRNA complex, actin-mediated cell contraction, or actin filament-based movement.