Identification of nuclear valosin-containing-protein-like as a target of anti-nuclear autoantibodies in systemic sclerosis

Abstract

Objective: To identify the target antigen of an anti-nuclear autoantibody (ANA) from a patient with a suspected systemic autoimmune disease and to study the autoantibody's clinical association.

Methods: The index patient serum was screened for autoantibodies using indirect immunofluorescence assay (IFA) and line blots (membrane strips coated with parallel lines of different purified antigens). Immunoprecipitation with fixed HEp-2 cells followed by SDS-PAGE and MALDI-TOF mass spectrometry was used to identify the autoantigen, which was verified by competitive inhibition experiments, recombinant HEK293 cell-based IFA, and Western and line blots based on the recombinant antigen. The prevalence of autoantibodies against this antigen was studied in 693 patients with systemic autoimmune rheumatic diseases (SARD) and 150 healthy controls.

Results: The index patient serum displayed a homogeneous nucleolar staining pattern on HEp-2 cells and monkey liver by IFA but did not react with 27 known nuclear antigens. Nuclear valosin-containing-protein-like (NVL) was identified as the ANA target antigen. Preincubation with recombinant NVL abolished the reactivity of the patient serum with HEp-2 cells in IFA. Additionally, the patient serum reacted with recombinant NVL in cell-based IFA and Western blot analysis, whereas sera from 15 healthy controls were nonreactive. Using line blots coated with recombinant NVL, anti-NVL autoantibodies were exclusively found in four out of 378 patients with systemic sclerosis, but neither in 315 patients with other SARD nor in 150 healthy controls.

Conclusion: These findings indicate that autoantibodies against NVL may be a suitable marker to help narrowing the serological gap in systemic sclerosis.

Keywords: ANA; NVL; anti-nuclear autoantibodies; autoimmune disease; biomarker; diagnosis; nuclear valosin-containing-protein-like; systemic sclerosis.

Figure 1

Identification and verification of nuclear valosin-containing-protein-like (NVL) as target of anti-nuclear autoantibodies. (A) Indirect immunofluorescence assay (IFA) with index patient serum (1:1000) on HEp-2 cells (top) and monkey liver (bottom), stained with anti-human-IgG-Alexa488 (1: 500, green) as secondary antibody and To-Pro-3 (1:2000, blue) as nuclear counterstain. Shown are single channel images (left, DNA only) and merged two-color images (right, DNA and patient serum). Magnification 630x. (B) Immunoprecipitates of serum from index patient or healthy control (HC) and HEp-2 cell lysate, stained with Coomassie after SDS-PAGE. The marked band was selected for mass spectrometry analysis. (C) Competitive inhibition experiments. Index patient serum (1:1000) was incubated with the 1:10 diluted purified recombinant NVL antigen (left) or control buffer (right) prior to application in IFA with HEp-2 cells. Anti-Human-IgG-Alexa488 (1: 500) was used as secondary antibody and To-Pro-3 (1:2000) was applied as nuclear counterstain. Shown are merged two-color images. Magnification 200x. (D) IFA with index patient serum (1:1000) on NVL-transfected (left) or control-transfected (right) HEK293 cells. Anti-Human-IgG-Alexa488 (1: 500) was used as secondary antibody and To-Pro-3 (1:2000) was applied as nuclear counterstain. Shown are merged two-color images. Magnification 200x. (E,F) Western blot of purified His-tagged NVL antigen incubated with anti-His tag antibody (1:2000), index patient serum (1:200), 15 sera from healthy controls (1:200) (E) or sera (1:200) from anti-NVL-positive patients with systemic sclerosis (SSc) (F). Anti-mouse-IgG-AP (1:2000) or anti-human-IgG-B/E-AP (1:10) were used in secondary incubation.

Figure 2

Tabular presentation of representative nuclear valosin-containing-protein-like (NVL) line blot strips after incubation with the anti-NVL-positive index patient serum, four anti-NVL-positive (SSc1-4) and five -negative (SSc5-9) sera from patients with systemic sclerosis, and five samples each of anti-NVL-negative disease controls (SLE1-5) and healthy controls (HC1-5). On each blot strip, the rightmost membrane chip was coated with a line of purified recombinant human NVL that becomes a purple band if a sample contains NVL-specific autoantibodies. Software-based evaluation was used to analyze and interpret the bands’ signal intensity. Correctly performed incubation and correctly used conjugate are indicated by a positive reaction of the serum control band (IgG) and of the conjugate control band (AGM), respectively.