Dermatan Sulfate Is a Potential Master Regulator of IgH via Interactions with Pre-BCR, GTF2I, and BiP ER Complex in Pre-B Lymphoblasts

Dermatan sulfate (DS) and autoantigen (autoAg) complexes are capable of stimulating autoreactive CD5+ B-1 cells. We examined the activity of DS on CD5+ pre-B lymphoblast NFS-25 cells. CD19, CD5, CD72, PI3K, and Fas possess varying degrees of DS affinity. The three pre-BCR components, Ig heavy chain mu (IgH), VpreB, and lambda 5, display differential DS affinities, with IgH having the strongest affinity. DS attaches to NFS-25 cells, gradually accumulates in the ER, and eventually localizes to the nucleus. DS and IgH co-localize on the cell surface and in the ER. DS associates strongly with 17 ER proteins (e.g., BiP/Grp78, Grp94, Hsp90ab1, Ganab, Vcp, Canx, Kpnb1, Prkcsh, Pdia3), which points to an IgH-associated multiprotein complex in the ER. In addition, DS interacts with nuclear proteins (Ncl, Xrcc6, Prmt5, Eftud2, Supt16h) and Lck. We also discovered that DS binds GTF2I, a required gene transcription factor at the IgH locus. These findings support DS as a potential master regulator of IgH in pre-B cells at protein and gene levels. We propose a (DS•autoAg)-autoBCR dual signal model in which an autoBCR is engaged by both autoAg and DS, and, once internalized, DS recruits a cascade of molecules that may help avert apoptosis and steer autoreactive B cell fate. Through its affinity with autoAgs and its control of IgH, DS emerges as a potential key player in the development of autoreactive B cells and autoimmunity.


Introduction
Autoimmunity, an immune response against the body self, is an intriguing phenomenon.
Autoimmunity is caused by autoreactive lymphocytes and/or autoantibodies that target autoantigens (autoAgs) present in single or multiple tissue types throughout the body, leading to over 80 recognized types of autoimmune diseases, ranging from systemic diseases such as lupus and rheumatoid arthritis to organ-specific diseases such as type 1 diabetes. Autoimmune phenomena are also strongly linked to various malignancies such as lymphoma and leukemia.
Despite extensive research, understanding how autoreactive lymphocytes survive rounds of immune editing and elimination has remained largely elusive. Although it is now accepted that autoreactive cells are positively selected by autoAgs, the underlying mechanism is unclear.
Furthermore, several hundred autoAgs have been identified across seemingly unrelated tissue locations or biologic functions. Yet these different autoantigens can all elicit a similar autoimmune response such as clonal expansion of autoreactive cells or production of specific autoantibodies. Better understanding of these processes will greatly advance the field of autoimmunity.
We have proposed that the glycosaminoglycan dermatan sulfate (DS) is a key missing player in autoimmunity in that (i) self-molecules with affinity to DS have a high propensity to be autoAgs and (ii) DS•autoAg complexes work in concert to stimulate autoreactive B cells (1, 2). In our initial study, we had injected mice with various glycosaminoglycans, including DS, heparan sulfate, chondroitin sulfates A and C, hyaluronic acid, and heparin, and found that DS is the most potent in inducing arthritis in mice (3). When tested in vitro with mouse splenocytes, DS is found to be the most potent in stimulating CD5+ B (B-1) cell proliferation and, moreover, DS exerts unique affinity to autoantigens from apoptotic cells, which prompted our hypothesis that DS and autoAgs form complexes and co-operate to stimulate B-1 cells (1). We then tested clinical autoantibody standard sera and sera from patients with autoimmune diseases such as lupus and Sjögren syndrome and demonstrated that DS affinity enabled the identification of many more autoAgs than current clinical tests are able to (2). Based on our DS•autoAg affinity hypothesis, we have thus far identified over 200 potential protein autoAgs from cells lines and murine liver and kidney tissues (2,4,5).
When autoAg•DS complexes encounter B cells expressing autoreactive B cell receptors (autoBCRs), we hypothesize that, while autoAgs engage autoBCRs, DS must engage other signals to ensure successful B-1 cell activation leading to further proliferation and differentiation.
Neither autoAgs nor DS alone can recruit a sufficient signaling network, as autoAg-autoBCR engagement without co-stimulating factors generally leads to deletion or anergy of the autoreactive cells. In our search for DS receptor(s) in autoreactive B-1 cells, we investigated CD5+ pre-B lymphoblast NFS-25 cells, a cell line derived from a spontaneous murine lymphoma (6). The pre-B stage is a crucial checkpoint for positive vs. negative regulation of autoimmunity and for development of mature autoreactive B cells. Precursor BCRs (pre-BCRs) are considered poly-and self-reactive, resembling polyreactive autoBCRs (7). Pre-BCRmediated signaling has been implicated in positive selection of pre-B cells, proliferation, survival, IgH allelic exclusion, and IgH repertoire selection (8). The pre-BCR is composed of a µ heavy chain (µH or IgH µ) and a germ line-encoded surrogate light chain made of λ 5 and VpreB (9).
Intriguingly, the pre-BCR shapes the IgH repertoire and appears to select particular IgH chains, such as those that express an H-CDR3 with positively charged amino acid residues such as arginines (10,11). Without any bias toward any particular signaling molecule, we carried out a series of experiments and discovered several lines of evidence to support that IgH is a critical DS receptor in a potential dual (DS•autoAg)-autoBCR signaling pathway and that DS is a potential master regulator of IgH.

DS activity on NFS-25 cell proliferation and apoptosis
In our previous study, when primary mouse spleen cells were cultured with DS, CD5+ B-1 cells were found to be specifically stimulated by DS to proliferate (1). To test whether a similar effect   holds for CD5+ pre-B cells, we cultured NFS-25 cells with DS, B-cell mitogen lipopolysaccharide   (LPS), or medium only. Cell proliferation assays showed no significant difference between NFS-25 cells cultured with DS, LPS, or medium alone for 3 to 6 days. It is possible that these cells may have to be cultured with a much higher concentration of DS or LPS or for longer time periods to show detectable differences.
In a previous study, we had discovered that DS has strong affinity to apoptotic cells (12). We therefore examined whether this DS-affinity property is also true for NFS-25 cells. These findings are consistent with our previously reported affinity of apoptotic cells to DS (1).
We also tested whether DS could rescue CPR-induced apoptotic cells by co-culturing cells with DS and CPT, but DS did not increase the viability rates of CPT-treated cells.  1A). CD5 molecules display a range of DS affinities, with the majority having no affinity but some having low affinity. However, two bands of smaller molecular size were also detected by anti-CD5 in fractions with medium and strong affinity. These findings suggest that CD5 may exist in multiple isoforms, and only some isoforms may associate with DS. CD72 was detected mostly in the 0.4 M fraction, although a higher molecular size version was present in the unbound fraction.
To ensure that the DS-affinity fractionation is indeed due to specific DS interaction but not nonspecific binding to the resin, we carried out competitive elution with DS or heparin. NFS-25 protein extracts were loaded onto DS-Sepharose resins and eluted with DS or heparin (Fig. 1B).
The majority of proteins bound to DS-Sepharose were eluted with DS at concentrations of 0.15, 0.2, or 0.4 M, whereas higher concentrations of heparin were needed. Western blotting verified that CD19, CD5, and CD72 were eluted with lower concentration of DS than heparin. These results suggest that proteins identified by our DS-affinity fractionation bind to DS more specifically than to heparin, a structurally similar polyanionic glycosaminoglycan.
We also examined a number of other markers as potential DS receptors (Supplemental Fig. 2).
Phosphoinositide 3-kinase (PI3K) has been identified in the DS signaling pathway in mouse spleen cell cultures (13,14). We therefore tested its affinity to DS. PI3K was detected in both Our previous study had shown that DS can also stimulate CD5+ cell proliferation in cells from CD19-deficient mice (1). The CD5 protein only partially co-localizes with DS (Supplemental Fig.   3). Although the above tested B cell signaling markers were detected in DS-binding factions and the results are consistent with our previous findings from mouse spleen B-1 cells (1), these proteins likely associate with DS indirectly via other molecules. Therefore, we continued our search for DS receptors by other means.

DS trafficking in NFS-25 cells and accumulation in the ER
We Differential DS affinity of pre-BRC components IgH µ, λ

5, and VpreB
The ER-resident protein Grp78 is also known as immunoglobulin heavy chain binding protein (BiP). We investigated whether immunoglobulins were involved in DS interactions in the ER. We anti-CD19 did also not enhance, but rather slightly reduced, IgH secretion when compared to cells cultured with anti-CD19 alone. It is possible that a DS-interacting site and CD5 may be in close proximity to CD19. Consequently, addition of DS or anti-CD5 would partially block and decrease anti-CD19-induced stimulation.
Pre-BCRs expressed in pre-B cells are formed by pairs of IgH µ chains and surrogate light chains composed of λ 5 and VpreB proteins. In addition to IgH, we asked whether λ 5 or VpreB also possess affinity to DS. We fractionated NFS-25 protein extracts and looked for pre-BCR components by blotting with a pre-BCR antibody that recognizes all three components (15). All

Discovery of direct interaction between DS and GTF2I
Given that DS traffics from the cell surface to the ER and nucleus, there could be numerous additional molecules involved in the DS-pre-BCR signaling network. We, therefore, took an unbiased approach to identify DS receptors. NFS-25 cells were cultured with or without DSbiotin for 2 days, proteins were extracted from 50 million cells, and both insoluble and soluble portions were collected. Proteins were then pulled down with NeutrAvidin beads and compared side by side in SDS-PAGE gel lanes. No difference was detected in the insoluble portion, but two distinct bands at ~120 kDa molecular weight were detected in the soluble protein portion of cells cultured with DS-biotin (Fig. 4).
Both bands were sequenced by mass spectrometry. The top band identified a single protein with 46 peptide matches to GTF2I, which is impressive as mass spectrometry sequencing of protein bands from cellular extracts usually gives rise to many identifications. The bottom band also overwhelmingly identified GTF2I with 49 peptide matches (Fig. 4). A few additional proteins were also identified in the bottom band, including Uba1 (12 peptide matches), importin 7 (Ipo7, 4 peptide matches), ATP-citrate synthase (Acly, 3 peptide matches), kinesin heavy chain isoform 5A (Kif5a, 2 peptide matches), Ipo5 (2 peptide matches), and DNA ligase 1 (Lig1, 2 peptide matches). Western blotting also confirmed these protein bands as GTFT2I (general transcription factor II-I), a homolog of human protein TFII-I.

DS affinity protein network and IgH-associated ER complex
We reasoned that a potential DS receptor in a cellular molecular complex first needs to be accessible to allow DS association. Therefore, we attempted to tag accessible proteins in NFS- Mass spectrometry sequencing of these bands identified 40 proteins with a minimum of 6 peptide matches as a cut-off (Supplemental Table 1 algorithm applied to the densely connected network identified a single dominant complex among these DS-affine proteins, the IgH chain associated ER-localized multiprotein complex.

Discussion
In this paper, we provide several lines of evidence to support DS as a master The IgH repertoire is generally thought to be positively selected at the pre-BCR checkpoint (8).
The pre-BCR assesses the quality of IgH chains and tunes the repertoire by driving the preferential expansion and differentiation of cells with higher quality of IgH µ chains (18). In particular, the pre-BCR appears to favor IgH chains with a CDR3 region containing positively charged amino acid residues such as arginines (10,11). Approximately 9% of in-frame IgH µ transcripts in human pro-B cells encode unusual IgH that can be expressed on the cell surface in the absence of surrogate or conventional light chains, and these IgH chains demonstrate preferential use of certain VH genes and an increased number of positively charged amino acid residues within the CDR3 region (10). Given that DS displays a high density repeating negative charges (on average, one carboxylate and one sulfate per repeating unit), we speculate that DS binds IgH at the CDR3 region.
Stromal cell-associated heparan sulfate, which is structurally similar to DS, has been proposed to be a potential pre-BCR ligand, as binding between pre-BCR and stromal cells could be specifically blocked by heparin, heparitinase, or a sulfate inhibitor (19). Moreover, the binding required the unique λ 5 tail, which protrudes from the pre-BCR molecule at the same position where the CDR3 of a conventional light chain is located (19). We had tested heparan sulfate, heparin, and other glycosaminoglycans in our previous studies, but found DS to be the most potent glycosaminoglycan for stimulating B-1 cells (1, 3) and hence have focused on DS.
Among the three components of the pre-BCR, our study found that IgH µ possesses strong DS affinity and λ 5 weaker affinity (Fig. 3). It is possible that DS binds to a positively charged IgH CDR3 conformation that is enforced by the λ 5 tail or conventional light chain CDR3.
Although synthesis and assembly rate of pre-BCR and BCR components are comparable, the pre-BCR is controlled by a highly efficient ER retention mechanism, which only allows exit of a few percent of the complexes from the ER (20). Furthermore, this retention mechanism is restricted to IgH and not selective for the surrogate light chain, thus appearing to be inherent to pre-B cells. Pre-B lymphocytes and hybridomas derived from them synthesize IgH in the absence of light chains, and BiP binds the CH1 domain of the free IgH and thus retains it in the ER (21,22). BiP also binds light chains shortly after their translation, and newly synthesized heavy and light chains interact sequentially with BiP and Grp94 in the ER (23,24). BiP and other ER molecules strictly control the processing and assembly of immunoglobulins, and our current study identify DS as another critical player of the Ig-associated ER complex.
B cells synthesize large amounts of immunoglobulins, which makes these cells particularly sensitive to ER stress. When CHO cells were transfected with an IgH µ chain, expressed µ chain accumulated in the cells and formed stable complexes with BiP, and the synthesis of three ER stress proteins (BiP, Grp94, and ERp72) increased at both protein and mRNA levels (25). We have shown that DS accumulates in the ER and has affinity to these and other stress proteins.
Our study shows, for the first time, GTF2I protein as a direct DS-interacting partner in pre-B cells. The GTF2I gene is well known for its association with Williams-Beuren syndrome and supravalvular aortic stenosis. Amazingly, recent large-scale genome-wide association studies have identified close association of the GTF2I gene with various autoimmune diseases.
Analysis of 556,134 autosomal SNPs in 542 cases and 1,050 controls followed by validation with 1,303 cases and 2,727 controls identified GTF2I at 7q11.23 (rs117026326) as a susceptibility locus for primary Sjögren syndrome (26). High-density genotyping of immunerelated loci with replication in 4,478 SLE cases and 12,565 controls from six East Asian cohorts identified GTF2IRD1-GTF2I at 7q11.23 to be the most significant locus associated with SLE (27). Variants at the GTF2IRD1-GTF2I locus have been identified as a susceptibility locus shared by SLE (27), primary Sjögren syndrome (26), rheumatoid arthritis (28), and myasthenia gravis (29). Single nucleotide polymorphisms (SNPs) located in the GTF2I-NCF1 region (rs73366469 (GTF2I), rs117026326 (GTF2I), rs80346167(GTF2IRD1) and rs201802880 (NCF1)) have strong association with SLE, Sjögren syndrome, rheumatoid arthritis, and ANCAassociated vasculitis (30). SNPs at rs117026326 are also associated with neuromyelitis optica spectrum disorder (31). GTF2I gene mutations occur at significantly higher frequencies in thymoma patients with myasthenia gravis than those without (29). GTF2I is required for optimal induction of GRP78/BiP and others in the ER stress response (39,40). When cells experience ER stress, the transcription of a family of glucose-regulated protein (GRP) genes which encode various ER chaperones is induced. The GRP promoters contain multiple copies of ER stress response element (ERSE), consisting of a unique CCAAT(N 9 )CCACG tripartite structure with N 9 being a GC-rich sequence of 9 bp that is conserved across species (40). GTF2I isoforms bind directly to the ERSEs of grp78 and ERp72 (PDIA4) promoters, and the stimulation of ERSE-mediated transcription by GTF2I requires consensus tyrosine phosphorylation sites on GTF2I and the ERSE GGC sequence motif (40).
Our study also identified 20 nuclear proteins, which are likely involved in GTF2I associated gene transcription activities. In addition, LCK is identified as a DS-associated protein.
Although typically regarded as a T-cell specific tyrosine kinase, LCK is an important mediator of BCR signaling in B-cell chronic lymphocyte leukemia (B-CLL) and found at significant levels in CD5+ B-1 cells (12,42). In mice, LCK is also expressed by CD5+ B-1 cells and involved in modulating BCR signal transduction in B-1 cells (43,44).
In our previous studies, we have demonstrated that DS has strong affinity to over 200 autoAgs and proposed that autoAgs and DS form macromolecular complexes and cooperate to stimulate autoreactive B cells (1,2,4,5). Therefore, we have been searching for DS receptors in autoreactive B cells. In this study, we identified IgH µ of the pre-BCR as a novel DS receptor.
Pre-BCRs resemble polyreactive autoBCRs in their capability of recognizing multiple autoAgs (7). Autoreactive B-1 cells differ from conventional B-2 cells in their responses to BCR signaling.
While binding of foreign antigens to BCRs on B-2 cells leads to cell activation, repeated encounters with autoAgs switch B-1 cells either into an anergic state or lead to apoptosis.
We propose a model of (DS•autoAg)-preBCR or (DS•autoAg)-autoBCR dual signaling that activates autoreactive B cells (Fig. 6). In this model, both autoAg and DS cooperate and bind simultaneously to different parts of the autoBCR. Once internalized with the BCR complex, DS recruits a network of additional partners that aids initial B-1 cell activation to proceed to the next stages of B cell development.
In summary, this study provides several lines of evidence that support DS as a potential master regulator of IgH in pre-B cells. DS interacts directly with IgH on the cell surface, with IgHassociated protein processing complexes in the ER, and with the GTF2I transcription factor that is required for IgH transcription. Through its affinity with autoAgs and its control of IgH, DS emerges as a potential key player in the development of autoreactive B cells and autoimmunity.

Materials and Methods
Cell culture

DS-affinity fractionation
DS-Sepharose resins were prepared by covalently linking DS (Sigma-Aldrich) to EAH Sepharose 4B resins (GE Healthcare) as previously described (1, 2). Aliquots of about 5.5 mg of protein extract in 1 ml of 10 mM phosphate buffer (buffer A, pH 7.4) was mixed with 0.35 ml of DS-Sepharose resins and incubated at 4 °C for 3 hours. After removing the buffer, the resins were washed three times with 1 ml buffer A to remove unbound proteins. Proteins bound to resins were then eluted stepwise and sequentially with 0.2, 0.4, 0.6, 1.0 M NaCl in buffer A, corresponding to proteins with no, low, medium, to high DS affinity, respectively. For each salt elution, resins were mixed with 1.0 ml of elution buffer for 5 min, then the eluate was collected, and each elution step was repeated three times. Eluted fractions were desalted and concentrated in 5 kDa cut-off Vivaspin centrifugal filters (Sartorius).

Competitive elution with DS and heparin
Proteins were extracted from NFS-25 cells and loaded onto DS-Sepharose resins as described above. After washing with 10 mM phosphate buffer three times, the resins were divided into two aliquots. In one aliquot, proteins were eluted sequentially with 0.

DS-biotin isolation of GTF2I
DS-biotin was synthesized as previously described (1