title: Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system.
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.
authors.affiliation: Recopharma AB, S-141 57 Huddinge, Sweden. anki.gustafsson@recopharma.com
authors.forename: Anki
authors.initials: A
authors.lastname: Gustafsson
authors.forename: Magnus
authors.initials: M
authors.lastname: Sjöblom
authors.forename: Lena
authors.initials: L
authors.lastname: Strindelius
authors.forename: Tomas
authors.initials: T
authors.lastname: Johansson
authors.forename: Tilly
authors.initials: T
authors.lastname: Fleckenstein
authors.forename: Nathalie
authors.initials: N
authors.lastname: Chatzissavidou
authors.forename: Linda
authors.initials: L
authors.lastname: Lindberg
authors.forename: Jonas
authors.initials: J
authors.lastname: Angström
authors.forename: Ulrika
authors.initials: U
authors.lastname: Rova
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism*; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D009077:Mucins; C084882:P-selectin ligand protein; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D008358:Mannose