Tomas Johansson

Folktandvården Stockholm AB, Tukholma, Stockholm, Sweden

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Publications (4)12.4 Total impact

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    ABSTRACT: Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/-B quantification and specificity determination, or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered CHO cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains.Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or -T2 and the A or B gene-encoded α1,3GalNAc-T or α1,3Gal-T, respectively. Selected clones with the correct glyco-phenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography - mass spectrometry was used to characterize released O-glycans.Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, while conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps.The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in ELISA or as affinity matrices in IA columns is explored.
    Glycobiology 02/2013; 23(6). DOI:10.1093/glycob/cwt011 · 3.15 Impact Factor
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    ABSTRACT: Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented. OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays. Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect. Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.
    PLoS ONE 10/2012; 7(10):e46959. DOI:10.1371/journal.pone.0046959 · 3.23 Impact Factor
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    ABSTRACT: Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta- and hexasaccharides being the predominant ones. α1,2- and α1,3-linked, but not α1,6-linked, mannose residues were detected by (1)H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG(2b) to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG(2b) expressed in P. pastoris carried O-glycans mainly comprised of α-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.
    Glycobiology 04/2011; 21(8):1071-86. DOI:10.1093/glycob/cwr046 · 3.15 Impact Factor

  • Journal of Biotechnology 10/2008; 136. DOI:10.1016/j.jbiotec.2008.07.649 · 2.87 Impact Factor