Dear
Madam or Sir,
In the meantime the "Method for proving antibodies in body fluids via an immune reaction with glycoprotein 2 (GP2) from zymogen granula of the pancreas for a differential diagnosis of inflammable intestinal illnesses and chronic pancreatitis" has been filed with the EPO (European Patent Application EP1950222).
With our newsletter special we would like to present an article that substantiates that "Glycoprotein2 (GP2), the major zymogen granule membrane glycoprotein, is the autoantigen of pancreatic antibodies in Crohn's disease". On behalf of our readers we very much like to thank the authors Dr. Dirk Roggenbuck (Dahlewitz, Germany), Professor René-Louis Humbel (Luxembourg), and Dr. Karsten Conrad (Dresden, Germany).
Sincerely,
Your DIARECT Team
Dirk Roggenbuck, René-Louis Humbel, Karsten Conrad
Crohn’s disease (CD) and ulcerative colitis (UC) are the two most frequently occurring inflammatory bowel diseases (IBD) in Caucasians. In general, both diseases affect as many as one in 250 individuals in developed countries. The incidence of CD is estimated at 5.6 per 100.000 individuals in one year in Europe. The aetiopathogenesis of CD has not yet been fully elucidated. First clinical manifestation of CD can occur relatively early at the age around 30 years, and the incidence of carcinoma is increased in patients with Crohn colitis after extended disease.
Mucosal inflammation in CD appears to occur when dysregulation of the immune system leads to an imbalance between tolerance to commensal microbiota or food-derived antigens and immunity to pathogens. It has been shown that autoimmune mechanisms play a role in the development of CD, and exocrine pancreas autoantibodies (PAB) are disease-specific in CD patients. In accordance with earlier studies, PABs, first described by Stöcker et al. using indirect immunofluorescence (IIF) in 1984, occur in up to 31% of all patients with CD. Also PABs have been detected in 68% of Crohn’s disease patients with extraintestinal complications such as idiopathic chronic pancreatitis. However, the target antigens and the underlying pathways that induce humoral autoimmunity in CD have not been sufficiently identified.
Recently, Roggenbuck et al. demonstrated for the first time that GP2 is the major autoantigenic target recognized by CD-specific PAB. In addition to IgG and IgM PAB isotypes, IgA pancreatic autoantibodies have also been detected in CD patients. Recently, IgA autoantibodies to GP2 have been identified in PAB-positive CD sera.
In IIF assays, PABs stain different structures of the exocrine pancreas and are accordingly divided into two subtypes: Subtype I PABs exhibit the typical extracellular drop-like staining pattern in the acinar lumen of pancreatic tissue sections and subtype II PABs lead to a speckled staining of the cytoplasm of pancreatic acinar cells. This is consistent with the localization of GP2 in the intercellular zymogen granules and in the pancreatic ducts after its release together with zymogens, respectively. Almost all subtype II PAB positive sera demonstrated PAB I reactivity in IIF. In contrast to recent data, Stöcker et al. reported that antibodies to GP2 only represent subtype I PAB revealing the extracellular drop-like staining whereas antibodies to CUZD1 are responsible for the speckled cytoplasmic staining of subtype II PAB.
Glycoprotein 2 is a highly glycosylated 78-kDa protein with N-linked carbohydrates. Glycoprotein 2 accounts for up to 40 percent of all zymogen granule (ZG) membrane proteins in pancreatic acinar cells and is linked to the ZG membrane via a glycosyl phosphoinositol (GPI) anchor. The membrane-bound and the free forms of GP2 are encoded by a single gene, which has been assigned to human chromosome band 9q21.11 to q21.2 by in situ hybridization.
Upon hormonal or neuronal stimulation of the pancreas, GP2 is transported to the apical compartment of acinar cells, from which it is released together with zymogens into the pancreatic duct. As a self-binding glycoprotein, GP2 forms soluble aggregates in pancreatic juice after cleavage. The physiological function of GP2 is still unknown. It has been suggested that Glycoprotein 2 influences granule formation by interacting with syncollin, zymogen granules ZG16p and ZG46p, and proteoglycans in a submembranous matrix. However, in a GP2-deficient mouse model, GP2 is not essential for exocrine secretion and zymogen granule formation.
Contradictory to data describing GP2 as a pancreas-specific protein, GP2 has also been shown to be specifically synthesized in murine and, recently, human M cells of Peyers’ patches (PP). Furthermore, GP2 mRNA transcription was detected in enterocytes of colon biopsies of CD patients and was significantly higher compared to UC patients. Peyers’ patches, which are particularly abundant in the distal part of the ileum, appear to be potential sites of the inflammatory onset in CD. Therefore, autoimmunity to GP2 can theoretically result in targeting of the intestine, the organ mainly affected by CD.
Interestingly, the GP2 amino acid sequence exhibits a similarity to Tamm-Horsfall protein (THP) or uromodulin. Tamm-Horsfall protein, also a GPI-anchored 85-KDa glycoprotein, is synthesized in cells of the ascending limb of the loop of Henle. Both glycoproteins contain a so-called D8C domain comprising 130 amino acids with eight conserved cystein residues, which is furthermore found only in liver-specific LZP. Interestingly, GP2 and THP possess, like the CUZD1 protein, a zona pellucida domain.
Although it is the most abundant urine protein, the function of THP remains unclear. However, defective THP synthesis has been shown to increase the susceptibility of mice to urinary tract infections. THP binds to uropathogenic type1 fimbriated Escherichia coli and prevents bacteria from interacting with urothelial (uroplakin) receptors. GP2 also binds a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. No specific pathogenic species has been associated with CD, but high concentrations of mucosal microbes, especially adhesive bacteria, have been detected in CD patients. Furthermore, there is an increased risk for development of IBD after gastrointestinal infections.
Tamm-Horsfall protein has also been reported to be a regulatory factor of innate and adaptive immunity of the urinary tract by interacting with toll-like receptor 4 (TLR-4). TLR-4 is also strongly up-regulated in enterocytes of CD patients. Remarkably, GP2 expression appears to be induced in the targeted tissue of patients with CD. Furthermore, GP2 was identified as a transcytotic receptor on M cells of PP for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Therefore, GP2 may have a similar effect in modulating innate and adaptive immunity.
A novel enzyme-linked immunosorbent assay (ELISA) for the detection of GP2 autoantibodies was evaluated employing recombinant GP2 expressed in the baculovirus system (Diarect AG, Germany). Autoantibody reactivity to GP2 and exocrine pancreas tissue (PAB) were assessed in PAB-positive CD patients (n=20), ulcerative colitis (CU) patients (n=20, PAB negative), and blood donors (n=50) by ELISA and IIF.
Nineteen of 20 PAB-positive CD patients tested positive in the novel ELISA. In contrast, no CU patient and one of 50 blood donors were positive. The anti-GP2 positive blood donor sample also displayed positive staining for PAB in IIF.
In summary, GP2 is a major and specific autoantigen of PAB in CD. Furthermore, GP2 was detected in human colon biopsies at the site of inflammation in CD and M cells of PP, the prospective onset site of inflammation. Further investigation of the role of GP2 in the aetiopathogenesis of CD is warranted. This novel autoantigen with its remarkable transcytotic receptor function may aid the understanding of IBD aetiology and help to resolve the remaining mysteries of IBD.
References
Aulinger-Stöcker K., Probst C, Komorowski L. Detection of Crohn’s disease specific autoantigens against exocrine pancreas by IIF using the newly identified proteoglycans CUZD1 and GP2 as recombinant target antigens. UEGW 2008, abstract.
Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet 2007;369:1627-31.
Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem 2006;52:171-81.
Hase K, Kawano K, Nochi T, et al. Uptake through glycoprotein 2 of FimH1 bacteria by M cells initiates mucosal immune response. Nature 2009;462:226-31.
Roggenbuck D, Hausdorf G, Martinez-Gamboa L, et al. Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn’s disease. Gut 2009;58:1620-8.
Stöcker W, Otte M, Ulrich S, et al. Autoantikörper gegen exokrines Pancreas und gegen intestinale Becherzellen in der Diagnostik des Morbus Crohn und der Colitis ulcerosa. Dtsch Med Wochenschr 1984;109:1963-69.
Wong SME, Lowe AW. Sequence of the cDNA encoding human GP-2, the major membrane protein in the secretory granule of the exocrine pancreas. Gene 1996;171:311-2.
DIARECT has produced a full-length human GP2 (human pancreatic secretory granule membrane major glycoprotein) in the baculovirus/Sf9 expression system. Preliminary test results have been achieved by use of the antigen in EIA systems (for details please see the article above).
Technical data:
Figure 1. GP2 was expressed in and purified from baculovirus-infected insect cells. Samples of lot 96B01 were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue or transferred to a PVDF membrane and probed with Penta-His antibody (Qiagen, Hilden).
Figure 2. Electrophoretic analysis of GP2 from lots 1468 and 96B01. Human recombinant protein was expressed in and purified from baculovirus-infected insect cells. The analysis shows the superior quality and lot-to-lot consistency of two independent productions.
DIARECT offers the most complete panel of recombinant targets for autoantibody testing. We are focused on our customer's success and committed to the highest level of quality in the field of assay components for diagnostic assays. It is our aim to provide our customers with a level of quality and service that consistently meets and exceeds their expectations.
Diagnostic use of Cat.No. 19600 GP2 in commercial test kits is protected by a patent; please contact Dr. D. Roggenbuck, Generic Assays, 15827 Dahlewitz, Germany - www.genericassays.com - in advance.
7th International Congress on Autoimmunity
Date: May 5 – 9, 2010
Location:
Ljubljana, Slovenia
12. Autoimmuntage in Bernau
Date: May
20 – 21, 2010
Location: Bernau, Germany
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