Expression and purification of recombinant M-Pol I from Saccharomyces cerevisiae with α-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 α-1,6 polymannose backbone attached to a Man8–10(GlcNAc)2 core. The backbone contains branches of α-1,2 mannose residues, terminated with α-1,3 mannose and decorated with α-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the α-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 cerevisae, 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 α-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 α-mannosidases revealed that the linkage formed is exclusively α-1,6. No α-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.