Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

End, Caroline; Lyer, Stefan; Renner, Marcus; Stahl, Cordula; Jutta, Ditzer
,; Holloschi, Andreas; Kuhn, Hella-M; Flammann, H.T.; Poustka, Annemarie; Hafner, Mathias; Mollenhauer, Jan; Kioschis, Petra

doi:10.1016/j.pep.2005.02.010

Cited by 28 publications

(30 citation statements)

References 36 publications

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“…They were preincubated in droplets of 50 ml mTBM supplemented with 0, 0.01, 0.1, 1, 10, or 100 mg/ml human recombinant DMBT1 (hrDMBT1) for 30 min in an atmosphere of 5% CO 2 in air at 39 8C in 100% humidity. The hrDMBT1 was kindly provided by Prof. C Robbe-Masselot from the University of Lille, France (End et al 2005, Rossez et al 2011. Owing to the low availability of equine ovaries in abattoirs and therefore of Oviductal DMBT1 is involved in fertilization equine oocytes for experiments, the effect of pre-incubation of equine oocytes with hrDMBT1 was not tested.…”

Section: Pre-incubation With Human Recombinant Dmbt1mentioning

confidence: 99%

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Ambruosi¹,

Accogli²,

Douet³

et al. 2013

Reproduction

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Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

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Section: Pre-incubation With Human Recombinant Dmbt1mentioning

confidence: 99%

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Ambruosi¹,

Accogli²,

Douet³

et al. 2013

Reproduction

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“…Detailed protocols on molecular cloning, the generation of stably transfected cell lines, and the purification of recombinant human DMBT1 (rhDMBT1) have been published elsewhere (34). Briefly, the largest known DMBT1 open reading frame, corresponding to the DMBT1/8kb.2 variant (EMBL accession number AJ243212), was cloned into the pT-Rex-DEST30 vector (Invitrogen Life Technologies) under the control of a tetracycline-inducible promoter.…”

Section: Recombinant Expression and Purification Of Human Dmbt1mentioning

confidence: 99%

Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion

Rosenstiel

Sina

End

et al. 2007

The Journal of Immunology

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158

163

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Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.

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“…Salivary DMBT1 (DMBT1 SAG ) was obtained from parotid saliva of a healthy volunteer and was purified essentially as previously described [20]. Expression and purification of hrDMBT1 were performed as previously described in detail elsewhere [40]. Concentrations of purified proteins were determined with the Micro BCA Protein Assay Reagent Kit from Pierce using bovine serum albumin as standard.…”

Section: Purification Of Dmbt1mentioning

confidence: 99%

“…High-affinity microtiter plates (Microlon Greiner-F, Polysorp, Nunc) were coated with serial dilutions of the ligands to be tested in 100 mM carbonate buffer, pH 9.6 at 41C for 16 h. After three washes with TBS containing 0.1% v/v Tween 20 (TBS-T), DMBT1 (10 mg/mL, corresponding to 40 nM in TBS-T) was added to each well and incubated at 371C for 1 h. Detection of bound DMBT1 with the DMBT1-specific mouse monoclonal antibody Hyb213-06 (Antibody-Shop; dilution 1:10 000 in TBS-T) was performed as previously described in detail elsewhere [40].…”

Section: Binding Studiesmentioning

confidence: 99%

DMBT1 functions as pattern‐recognition molecule for poly‐sulfated and poly‐phosphorylated ligands

et al. 2009

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Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-jB activation and to the pathogenesis of Crohn's disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen-binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1-mediated bacterial aggregation via interaction with its bacterial-recognition motif. Competition and ELISA studies identify poly-sulfated and poly-phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose-response studies in Dmbt1 À/À and Dmbt1 1/1 mice utilizing the DSS-induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern-recognition molecule for poly-sulfated and polyphosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-jB activation. IntroductionCrohn's disease (CD) is one of the major subtypes of inflammatory bowel disease (IBD) and is thought to emerge through the complex interplay of various environmental and genetic factors. Nutritional compounds, bacterial pathogens, and the endogenous bacterial microflora have been considered as environmental factors contributing to the etiology of CD [1,2]. The putative involvement of bacteria is also reflected at the level of susceptibility genes for CD, of which some appear to exert important functions in innate mucosal immunity [1,3,4]. Genetic variants of the gene nucleotide-binding oligomerization domain 2/Caspase recruitment domain 15 (NOD2/CARD15) at the IBD1 locus, for example, have been shown to be associated with an increased risk for CD [5][6][7]. NOD2 encodes an intracellular pattern-recognition receptor with specificity for muramyldipeptide, and is thought to function in the sensing of intracellular bacteria [8]. Dysfunction of NOD2 may result in altered NF-kB signaling and attenuated expression of secreted anti-bacterial factors such as a-defensin-2 [9-11]. In addition, decreased copy numbers of the b-defensin-4 gene DEFB4 (HBD-2) at chromosome 8p23.1 have been reported to result in reduced DEFB4 expression levels and to confer an increased risk for CD [4].Recent studies demonstrated that deleted in malignant brain tumors 1 (DMBT1) is up-regulated in the inflamed mucosa of CD patients with normal NOD2 but not in those with the frequent CD-associated L1007fsinsC mutation in NOD2 [12,13]. The DMBT1 gene locates at human chromosome 10q26.13 and ...

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Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Cited by 28 publications

References 36 publications

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion

DMBT1 functions as pattern‐recognition molecule for poly‐sulfated and poly‐phosphorylated ligands

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