Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system

Glycobiology. 2011 Aug;21(8):1071-86. doi: 10.1093/glycob/cwr046. Epub 2011 Apr 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Carbohydrate Sequence
  • Cricetinae
  • Cricetulus
  • Lectins, C-Type / immunology
  • Lectins, C-Type / metabolism*
  • Mannose / chemistry
  • Mannose / metabolism
  • Mannose-Binding Lectins / immunology
  • Mannose-Binding Lectins / metabolism*
  • Membrane Glycoproteins / biosynthesis
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Molecular Sequence Data
  • Mucins / biosynthesis*
  • Mucins / chemistry*
  • Mucins / genetics
  • Mucins / metabolism
  • Pichia / genetics*
  • Receptors, Cell Surface / immunology
  • Receptors, Cell Surface / metabolism*
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics

Substances

  • Lectins, C-Type
  • Mannose-Binding Lectins
  • Membrane Glycoproteins
  • Mucins
  • P-selectin ligand protein
  • Receptors, Cell Surface
  • Recombinant Fusion Proteins
  • mannose receptor
  • Mannose