Human proteins with affinity for dermatan sulfate have the propensity to become autoantigens

Abstract

The mystery of why and how a small, seemingly disparate subset of all self molecules become functional autoantigens holds a key to understanding autoimmune diseases. Here and in a companion article in this issue, we show that affinity of self molecules to the glycosaminoglycan dermatan sulfate (DS) is a common property of autoantigens and leads to a specific autoreactive B-1a cell response. Autoimmune ANA/ENA reference sera react preferentially with DS affinity-fractionated cellular proteins. Studying patients with autoimmune diseases, we discovered patient-specific complex autoantigen patterns that are far richer and more diverse than previously thought, indicating significant pathological heterogeneity even within traditionally defined clinical entities, such as systemic lupus erythematosus. By shotgun sequencing of DS affinity-enriched proteomes extracted from cell lines, we identified approximately 200 autoantigens, both novel and previously linked to autoimmunity, including several well-known families of autoantigens related to the nucleosome, ribonucleoproteins, the cytoskeleton, and heat shock proteins. Using electron microscopy, we recognized direct interaction with dead cells as an origin of autoantigenic association of DS with self molecules. DS affinity may be a unifying property of the human autoantigen-ome (ie, totality of self molecules that can serve as functional autoantingens) and thus provides a promising tool for discovery of autoantigens, molecular diagnosis of autoimmune diseases, and development of cause-specific therapies.

Figure 1

A: Classic ANA/ENA autoantigens are characterized by affinity to DS. Proteome extracts from WIL2-NS cells were fractionated on DS-affinity columns; 10 μg of total proteins were loaded per lane. Gels were blotted with individual ANA or ENA reference serum (each gel is labeled with the nominal reference serum specificity). B: DS interacts with Scl-70∙DNA complexes. Left: ENA and ANA panels. Proteome extracts from WIL2-NS or HEp-2 cells were fractionated by DS affinity and blotted with two different Scl-70 reference sera. Note that smeared bands at high molecular weight were detected in DS-binding fractions. Right: For the ANA panel, WIL2-NS proteins eluted with 0.6 mol/L NaCl were treated with PNGase F, DNase I, or RNase A and analyzed by electrophoresis and using Western blot analysis with Scl-70 reference serum. Only after digestion with DNase I did the smeared bands disappear, whereas the band corresponding to Scl-70 increased in intensity, suggesting that the smear corresponds to Scl-70∙DNA complexes. Lane labels: T, total unfractionated extract; numbers above lanes indicate the molar concentration of NaCl used for the respective elution step.

Figure 2

Autoantigen profiling in autoimmune disease shows unexpected diversity. Proteins extracted from HEp-2 or WIL2-NS cells were DS-affinity fractionated (T, total unfractionated extract; numbers above lanes indicate the molar concentration of NaCl used for the respective elution step); 8 μg of total proteins were loaded per lane. Autoantigens were detected using Western blot analysis with individual patient serum (gels for patients 1 to 25 are shown). The ∼80-kDa band frequently appearing in not/weakly DS-binding (0.2 mol/L NaCl) fractions was identified as Ig γ-1 chain C (see Supplemental Figure S3D at http://ajp.amjpathol.org), which reacts with the secondary anti-IgG used for blotting and thus corresponds to a false positive band.

Figure 3

Direct association of DS with dead WIL2-NS cells. WIL2-NS cells were cultured with DS-biotin for 1 day to 3 days, and frozen sections of cells were labeled with anti-biotin gold particle conjugates (diameter, 20 nm) for examination by electron microscopy. A: DS (small dots) associated with dead cell debris (arrows) just outside a viable cell (visible to the left). B: Higher magnification showing DS association with cell debris. C: DS associated with fibrillar membrane-type cell debris. D: DS associated with dead cell blebs. Note part of a dead cell nucleus (left bottom corner). Scale bar = 500 nm (all panels).