Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease.
OBJECTIVE
The aetiopathogenesis of Crohn's disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn's disease, but the target antigens and the underlying pathways have not been sufficiently identified.


METHODS
Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn's disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies.


RESULTS
The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn's disease. PAB-positive sera from patients with Crohn's disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p<0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn's disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn's disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn's disease.


CONCLUSIONS
Anti-GP2 autoantibodies constitute novel Crohn's disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn's disease.
Roggenbuck
D
D
Institute of Immunology, Technical University Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
Hausdorf
G
G
Martinez-Gamboa
L
L
Reinhold
D
D
Büttner
T
T
Jungblut
P R
PR
Porstmann
T
T
Laass
M W
MW
Henker
J
J
Büning
C
C
Feist
E
E
Conrad
K
K
D000328:Adult; D000368:Aged; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D003093:Colitis, Ulcerative / Q000235:genetics / Q000276:immunology; D003106:Colon / Q000276:immunology; D003424:Crohn Disease / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D058851:GPI-Linked Proteins; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D008875:Middle Aged; D010179:Pancreas / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000276:immunology; D022142:Secretory Vesicles / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D055815:Young Adult
D001323:Autoantibodies; D001324:Autoantigens; C489339:GP2 protein, human; D058851:GPI-Linked Proteins; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins