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AGR2, an Endoplasmic Reticulum Protein, Is Secreted Into the Gastrointestinal Mucus

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AGR2, an Endoplasmic Reticulum Protein, Is Secreted Into the Gastrointestinal Mucus AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus Joakim H. Bergstro¨ m1, Katarina A. Berg1, Ana M. Rodrı´guez-Pin˜ eiro1,Ba¨rbel Stecher2, Malin E. V. Johansson1, Gunnar C. Hansson1* 1 Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden, 2 Max von Pettenkofer Institute for Hygiene and Medical Microbiology, LMU Munich, Munich, Germany Abstract The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr22/2 mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine. Citation: Bergstro¨m JH, Berg KA, Rodrı´guez-Pin˜eiro AM, Stecher B, Johansson MEV, et al. (2014) AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus. PLoS ONE 9(8): e104186. doi:10.1371/journal.pone.0104186 Editor: Jean-Luc Desseyn, Inserm, France Received March 16, 2014; Accepted July 11, 2014; Published August 11, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript. Funding: This work was supported by the Swedish Research Council, The Swedish Cancer Foundation, The Knut and Alice Wallenberg Foundation, IngaBritt and Arne Lundberg Foundation, Sahlgren’s University Hospital (LUA-ALF), Wilhelm and Martina Lundgren’s Foundation, Torsten och Ragnar So¨derbergs Stiftelser, The Sahlgrenska Academy, the National Institute of Allergy and Infectious Diseases (U01AI095473, the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH), German Research Foundation (DFG STE1971/4-1), and The Swedish Foundation for Strategic Research - The Mucus-Bacteria-Colitis Center (MBC) of the Innate Immunity Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction well as regulating cell proliferation [8,9]. The Xenopus and Salamander mechanisms are due to secreted forms of the AGR- The gastrointestinal tract is protected by mucus that is analogues, but the localization of the growth promoting effect in differently organized along the intestine [1]. In the small intestine mammals is less understood [9]. Recent studies using recombinant there is a single mucus layer, whereas mucus in the stomach and monomeric AGR2 suggested that it could bind on the outside of colon is double layered. In colon the inner mucus layer is cells suggesting that AGR2 could have an extracellular effect [10]. important for separating bacteria from the epithelium [2]. Defects Mature AGR2 is a small 154 amino acid protein with a single in this inner mucus layer allow bacteria to get in contact with the central Cys residue and a non-conventional endoplasmic reticu- epithelial cells, an event which can trigger an inflammatory lum (ER) retention motif (KTEL). The structure of AGR2 was reaction [3,4]. The MUC2 mucin forms the skeleton of the recently revealed and suggested that AGR2 was appearing as a intestinal mucus [4]. This gel-forming mucin is thus instrumental non-covalent dimer in the ER [10]. The Cys residue is part of the in maintaining a functional inner colon mucus layer, and its CXXS motif that is found in the large family of disulfide absence leads to severe colitis and cancer development [2,5]. This isomerases (PDI) and as many members of these proteins also have places the goblet cells, which biosynthesize the MUC2 mucin, in ER-retention signals, AGR2 have been suggested to act as a PDI the focus of understanding mucus. and being involved in controlling ER homeostasis [11]. Such a Anterior gradient 2 protein (AGR2) is part of the three function is supported by the observation that AGR2 can act as a membered AGR family first identified to be involved in control protein disulfide isomerase-like molecule important for MUC2 of the cement gland and brain development of Xenopus [6]. In biosynthesis [12]. To further study AGR2 function, several mice salamanders anterior gradient-related proteins Nag and Mag lines (Agr22/2) have been developed [12–14]. All of these animals induce the spectacular regeneration of lost vertebrate limbs [7]. In show alterations in different parts of the gastro-intestinal tract. mammalian breast tissue AGR2 expression is estrogen controlled As AGR2 has been suggested to be involved in the MUC2 and regulates mammary epithelial proliferation [8]. Thus AGR mucin biosynthesis we found it essential to further understand the proteins have growth promoting effects, a property that is linked to relation between AGR2 and MUC2 [12]. This is of further the role of AGR2 in cancer development and metastasis of several importance as AGR2 had been genetically linked to inflammatory tumor types [9]. AGR2 is proposed to act as a pro-oncogene as bowel disease susceptibility [15]. We thus decided to analyze the PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104186 AGR2 in Mucus interaction of AGR2 with the MUC2 mucin in more detail in vitro For construction of the DNA-plasmid encoding a version of using a cell culture model and in vivo in Agr22/2 mice. In the cell AGR2 without the KTEL sequence (AGR2 DKTEL), the Lys culture model we could not confirm direct covalent binding of residue in the terminal KTEL peptide was replaced by a STOP AGR2 to MUC2. We also observed that AGR2 was secreted when codon. pcDNA3.1D-AGR2 was PCR amplified with the forward its single Cys was removed (C81S). Lastly, we could show that the primer 59-CAAGTTGCTGTAGGAGCTCTTGTAGGGTCA- intestinal mucus contained a high concentration of secreted Agr2, AGAC-39 and the reverse primer 59-GTCTTGACCCTACAA- suggesting extracellular effects among the poorly understood GAGCTCCTACAGCAACTTG -39. The PCR conditions were functions of this protein. the same as for the AGR2 C81S. All generated plasmids were controlled by nucleotide sequencing after mutagenesis. Materials and Methods Tissue culture Generation of the pcDNA3.1-AGR2 plasmid CHO-K1 cells were obtained from the American Type Culture The full length cDNA sequence of the human AGR2 was PCR- Collection (CCL-61) and cultured in Iscove’s modified Dulbecco’s amplified from the RZPD clone IRATp970F1215D6 with the medium (Lonza) containing 10% (v/v) fetal bovine serum. The cell primers 59-CACCATGGAGAAAATTCCAGTGTC-39 and 59- line CHO-pSNMUC2-MG was cultured in the same medium CAATTCAGTCTTCAGCAACTTGAGAGC-39 to introduce a supplemented with G418 (250 mg/mL) [16]. Cells transfected with 59 CACC overhang, needed for directional cloning into the vector pcDNA3.1/Zeo-AGR2 were additionally kept in Zeocin (Life pcDNA3.1D/V5-His-TOPO (Life Technologies). Next, a stop Technologies) (250 mg/mL). codon was introduced in the vector after the AGR2 sequence by site-directed mutagenesis (QuickChange site-directed mutagenesis Transfection kit; Stratagene) using the primers 59- CTCAAGTTGCTGAA- Transient transfections were made in CHO-K1 cells or CHO- GACTGAATTGTAGGGTCAAGACAATTCTGCAG -39 and pSNMUC2-MG cells using to the Lipofectamine 2000. After 59-CTGCAGAATTGTCTTGACCCTACAATTCAGTCTTC- 48 hours the supernatant was collected. The cells were then AGCAACTTGAG-39 (mismatch is underscored). The PCR was incubated 30 min on ice with lysis-buffer containing Complete initiated at 95 C for 30 s, followed by 15 cycles (95 C for 30 s, u u EDTA-free protease inhibitors (Roche), and collected. 55uC for 1 min and 10 min at 68uC with a final elongation for 10 min at 68uC.The resulting vector was designated as SDS-PAGE and immunoblotting pcDNA3.1-AGR2 and encoded an untagged version of the human AGR2. For protein separation, samples were mixed with Laemmli sample buffer with or without 200 mM dithiothreitol (DTT), incubated for 5 min at 95 C and separated in a 3–15% Subcloning with pcDNA3.1/Zeo(+) u polyacrylamide gel by discontinuous SDS-PAGE using a 3% To make stable clones of CHO-psNMUC2-MG [16] and stacking gel [17]. The Precision Protein Standard (Bio-Rad) was AGR2, the plasmid pcDNA3.1-AGR2 was subcloned to be used as molecular marker. The separated proteins were Western- resistant against Zeocin. The AGR2-containing sequence was blotted onto polyvinylidene difluoride (PVDF) membranes (Im- excised from the pcDNA3.1-AGR2 vector with the restriction mobilon-PSQ Transfer Membrane, Millipore) using a Trans-Blot endonucleases BamH1 and Xba1,
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