Journal
GLYCOBIOLOGY
Volume 21, Issue 8, Pages 1071-1086Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwr046
Keywords
DC-SIGN; mannose-binding lectin; mannose receptor; mucin; Pichia pastoris
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Funding
- Recopharma AB
- Swedish Research Council/Medicine [K2011-65X-3031-01-6]
- County Councils of Stockholm and Vastra Gotaland (ALF)
- Marianne and Marcus Wallenberg Foundation
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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 alpha 1-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 alpha-mannosidases including an alpha 1,2- but not an alpha 1,6-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. alpha 1,2- and alpha 1,3-linked, but not alpha 1,6-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 alpha-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.
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