Targeting of active rat alpha 2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150 delta-carrier: toward synthesis of sLe(x)-decorated L-selectin ligands.
Interactions between selectins and their oligosaccharide-decorated ligands play a crucial role in the initiation of leukocyte extravasation. We have shown that synthetic multivalent sialyl Lewis x glycans inhibit strongly the adhesion of lymphocytes to endothelium at sites of inflammation. However, enzyme-assisted synthesis of these oligosaccharides si hampered by the lack of sufficient amounts of specific glycosyltransferases. We report here the construction of Saccharomyces cerevisiae strains expressing the soluble catalytic ectodomain of rat Gal(beta)1-3/4GlcNac alpha 2,3-sialyltransferase (ST3Ne) fused to the C-terminus of the hsp150 delta-carrier polypeptide. The hsp150 delta-carrier, which is an N-terminal fragmented of a natural secretory protein of yeast, is able to confer secretion-competence to several heterologous proteins, which otherwise remain in the yeast endoplasmic reticulum. The ST3Ne portion of the hsp 150 delta-ST3Ne fusion protein adopted an enzymatically active conformation and was N-glycosylated and disulfide-bonded. Hsp150 delta-ST3Ne was secreted with a half-time of about 7.5 min and remained intercalated in the cell wall, which covers the yeast plasma membrane. About 110 mU of sialyltransferase per litre was produced in 16 h. Whole live yeast cells were able to transfer sialic acid from CMP-NeuNAc to N-acetyllactosamine yielding alpha 2,3-sialyl-N-acetyllactosamine, as evidenced by paper chromatography, cleavage by linkage-specific sialidase, and NMR analysis. Our data suggest that yeast cells externalizing mammalian glycosyltransferases with the aid of the hsp150 delta-carrier could provide a source of enzymes for synthesis of valuable oligosaccharides.
Mattila
P
P
Institute of Biotechnology, University of Helsinki, Finland.
Joutsjoki
V
V
Kaitera
E
E
Majuri
M L
ML
Niittymäki
J
J
Saris
N
N
Maaheimo
H
H
Renkonen
O
O
Renkonen
R
R
Makarow
M
M
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002473:Cell Wall / Q000201:enzymology*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D019041:L-Selectin / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases / Q000378:metabolism*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D008024:Ligands; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D019041:L-Selectin; D012799:Sialyltransferases; D000117:Acetylglucosamine; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase