A practical approach for O-linked mannose removal: the use of recombinant lysosomal mannosidase.
The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris. However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris. Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.
GlycoFi, Inc. (a wholly owned subsidiary of Merck & Co., Inc.), Biologics Discovery, Merck Research Laboratories, 16 Cavendish Court, Lebanon, NH, 03766, USA.
Daniel
D
Hopkins
Sujatha
S
Gomathinayagam
Stephen R
SR
Hamilton
D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D006023:Glycoproteins; D011994:Recombinant Proteins; D008361:Mannosidases; D008358:Mannose