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

Abstract

Dermatan sulfate (DS) and autoantigen (autoAg) complexes are capable of stimulating autoreactive CD5+ B1 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 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.

Keywords: BiP; GTF2I; Ig heavy chain; autoimmunity; dermatan sulfate (DS); precursor BCR.

Figure 1

(A) Protein extracts from NFS-25 cells were fractionated with DS-affinity resins and blotted with specific antibodies. Protein lanes correspond to total unfractionated proteins (T) and fractions eluted with 0.2, 0.4, 0.6, and 1.0 M NaCl, respectively. (B) NFS-25 proteins loaded onto DS resins were eluted with step gradients of DS or heparin (left: Coomassie blue-stained SDS-PAGE) and blotted with specific antibodies (right), demonstrating competitive binding specificity between DS-resin-binding proteins and DS or heparin. DS and heparin concentrations are expressed as µmol/ml of disaccharide.

Figure 2

NFS-25 cells cultured with DS-AF568 (red) for various times were stained with anti-CD19 (green) and DAPI (blue). After 1 hour, trace amounts of DS were observed on some cell surfaces (white arrow). After 4 hours, DS association with small apoptotic cell bodies was observed (white arrows). From 8 hours on, DS starts to accumulate inside viable cells. At about 48 hours, intracellular DS reaches maximum intensity, with a majority in the ER and some also in the nucleus. Each panel shows four quadrants: green (upper left), red (upper right), blue (lower left), and merged (lower right) channels.

Figure 3

(A) NFS-25 cells cultured with DS-AF568 and stained with anti-Grp78/BiP confirming the DS-accumulating compartment as the ER. (B) NFS-25 cell proteins fractionated by DS affinity and blotted with anti-Grp78/BiP. (C) NFS-25 cell proteins fractionated by DS affinity and blotted with anti-preBCR, revealing the 3 components of preBCR, each of which has different DS affinity, with IgH µ having strongest affinity. (D) NFS-25 cells cultured with DS-AF568 and stained with anti-IgH µ showing co-localization on the cell surface and in the ER. Each panel shows four quadrants: green (upper left), red (upper right), blue (lower left), and merged (lower right) channels.

Figure 4

(A) Proteins extracted from NFS-25 cells cultured with DS-biotin (DSb) or without (CT, control) and analyzed by SDS-PAGE. The two arrows indicated two forms of GTF2I. (B) The identity of GTF2I is confirmed by blotting with anti-GTF2I. (C) Amino acid sequence of GTF2I (canonical isoform 1) with green regions highlighting peptides that were identified by mass spectrometric sequencing of the gel band indicated by the upper arrow in (A).

Figure 5

Protein-protein interaction network of the 40 biotin-tagged DS-binding proteins. Green: proteins involved in protein processing in the ER. Dark green: proteins involved in protein folding in the ER. Blue: nuclear proteins.

Figure 6

Proposed model of dual signaling by DS and autoAg in non-covalent (DS•autoAg)-autoBCR or (DS•autoAg)-pre-BCR complexes for activation of B1 cells.