title: Soluble expression of disulfide-bonded C-type lectin like domain of human CD93 in the cytoplasm of Escherichia coli.
abstract: CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2+was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).
authors.affiliation: Inserm, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, Plateforme CYROI, Sainte-Clotilde, F-97490, France; GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France.
authors.forename: Brice
authors.initials: B
authors.lastname: Nativel
authors.affiliation: GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France; Inserm, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, Plateforme CYROI, Sainte-Clotilde, F-97490, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Figuester
authors.affiliation: GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Andries
authors.affiliation: Inserm, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, Plateforme CYROI, Sainte-Clotilde, F-97490, France.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Planesse
authors.affiliation: Inserm, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, Plateforme CYROI, Sainte-Clotilde, F-97490, France.
authors.forename: Joël
authors.initials: J
authors.lastname: Couprie
authors.affiliation: GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France; UM134, Processus Infectieux en Milieu Insulaire Tropical (PIMIT), Université de La Réunion, INSERM1187, CNRS 9192, IRD 249, Plateforme CYROI, Sainte-Clotilde F-97490, France; Laboratoire de Biologie, LICE-OI, CHU de la Réunion, 1 allée des Topazes, 97400, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gasque
authors.affiliation: GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France; UM134, Processus Infectieux en Milieu Insulaire Tropical (PIMIT), Université de La Réunion, INSERM1187, CNRS 9192, IRD 249, Plateforme CYROI, Sainte-Clotilde F-97490, France. Electronic address: wildriss.viranaicken@univ-reunion.fr.
authors.forename: Wildriss
authors.initials: W
authors.lastname: Viranaicken
authors.affiliation: GRI, EA4517, Université de la Réunion, Saint-Denis F-97400, France; CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Iwema
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000235:genetics; D000072417:Protein Domains; D011951:Receptors, Complement / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D002118:Calcium; D004220:Disulfides; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011951:Receptors, Complement; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D050862:Oxidoreductases Acting on Sulfur Group Donors; C032402:complement 1q receptor; C526649:dsbC protein, E coli
keywords: C-type like domain (CTLD); CD93; Disulfide bond; DsbC; Erv1p; Inflammation