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

• 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/mIgG2b) carrying mostly O-glycans and, as a control, the a1-acid glycoprotein (AGP/mIgG2b) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. P. pastoris-produced PSGL-1/mIgG2b was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by a-mannosidases including an a1,2- but not an a1,6-mannosidase. Electrospray ionization - ion trap mass spectrometry (ESI-MS) confirmed the presence of O-glycans containing up to 9 hexoses with the penta- and hexasaccharides being the predominant ones. a1,2- and a1,3-linked, but not a1,6-linked, mannose residues were detected by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG2b to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG2b expressed in P. pastoris carried O-glycans mainly comprised of a-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.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

Nyckelord

Biokemisk processteknik

Biochemical Process Engineering

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)