Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with alpha-1,6 mannosylpolymerase activity.
Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 mannose and decorated with alpha-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the alpha-1,6 linked mannose backbone by the M-Pol I complex. Constructs encoding soluble portions of the M-Pol I subunits, Mnn9p and Van1p from Saccharomyces cerevisiae, were expressed in Pichia pastoris. Both subunits had to be expressed in the same strain to obtain the recombinant proteins. Recombinant M-Pol I was made only by the KM71 strain transformed with two vectors: one encoding Mnn9p and the other encoding Van1p. Soluble secreted M-Pol I was purified by sequential chromatography on DEAE-Trisacryl, GDP-Hexanolamine-Sepharose and Superdex 200. Characterization of the purified complex indicates that recombinant M-Pol 1 is a Mnn9p-Van1p heterodimer. Purified M-Pol I was active with alpha-1,6 mannobiose as acceptor and GDP-mannose as donor. HPLC identified five products confirmed to be 3-7 mannose residues long. Digestion with linkage-specific alpha-mannosidases revealed that the linkage formed is exclusively alpha-1,6. No alpha-1,2 mannosyltransferase activity, reported previously for M-Pol I immunoprecipitates from cell extracts was detected. These results provide further information on the role of M-Pol I in mannan biosynthesis.
Rodionov
Dmitry
D
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Romero
Pedro A
PA
Berghuis
Albert M
AM
Herscovics
Annette
A
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D008364:Mannosyltransferases; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
C086605:MNN9 protein, S cerevisiae; D008351:Mannans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C063125:VAN1 protein, S cerevisiae; D008364:Mannosyltransferases