Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution.
The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110-170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; n = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116-121 and 143-148. Indirect peptide ELISA analysis of the sera of patients with SLE (n = 88) revealed that ∼14% of patients had serum IgG reactivity to 116-121, while reactivity to 143-148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116-121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (n = 92). Competitive ELISA showed antibodies to 116-121 bind a common epitope in U1-70K (68-72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics.
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
David James
DJ
Haddon
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Justin Ansel
JA
Jarrell
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Vivian K
VK
Diep
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Hannah E
HE
Wand
b Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis , University of California at Berkeley , Berkeley , CA , USA .
Jordan V
JV
Price
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Stephanie
S
Tangsombatvisit
c Intel Corporation , Santa Clara , CA , USA .
Grace M
GM
Credo
d Department of Pediatrics, Division of Infectious Diseases , Stanford University School of Medicine , Stanford , CA , USA , and.
Sally
S
Mackey
d Department of Pediatrics, Division of Infectious Diseases , Stanford University School of Medicine , Stanford , CA , USA , and.
Cornelia L
CL
Dekker
e Department of Medicine , Division of Rheumatic and Autoimmune Diseases, University of Minnesota Medical School , Minneapolis , MN , USA.
Emily C
EC
Baechler
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Chih Long
CL
Liu
c Intel Corporation , Santa Clara , CA , USA .
Madoo
M
Varma
a Department of Medicine , Division of Immunology & Rheumatology, Stanford University School of Medicine , Stanford , CA , USA .
Paul J
PJ
Utz
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000276:immunology; D001323:Autoantibodies / Q000737:chemistry* / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D016022:Case-Control Studies; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D040081:Protein Array Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000276:immunology* / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry
D000596:Amino Acids; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D007074:Immunoglobulin G; C545845:M1 matrix protein, influenza B virus; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; D014763:Viral Matrix Proteins
RNP; SLE; Silicon-based peptide microarray; ribonucleoprotein; systemic lupus erythematosus