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The isolation and characterization of the gene encoding the glycosidase II alpha subunit of the industrially important fungus Trichoderma reesei is described. The gene encoding the glucosidase II alpha subunit in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product.

An engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans are described. This is done by inactivation of OCH1 and over expression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetyglucosaminyltransferase I and beta-1,4-hybrid-type N-linked oligosaccharide structure on its glycoproteins.

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with ?-mannosidase to produce in yeast a form of the Pompe disease enzyme ?-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of ?-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with nonhuman high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2- mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans. [ABSTRACT FROM AUTHOR]

The analysis of protein-linked glycans is of increasing importance,both in basic glycobiological research and during the productionprocess of glycoprotein pharmaceuticals. In many cases, the amountof glycoprotein available for typing the glycans is very low. This,combined with the high branching complexity typical for this classof compounds, makes glycan typing a challenging task. We presenthere methodology allowing the medium-throughput analysis of N-glycansderived from low picomole amounts of glycoproteins using the standardDNA-sequencing equipment available in any life sciences laboratory.The high sensitivity of the overall analytical process (from glycoproteinto results) is obtained using state-of-the-art deglycosylation procedurescombined with a highly efficient and reproducible novel postderivatizationcleanup step involving Sephadex G10 packed 96-well filterplates. Allsample preparation steps (enzymatic deglycosylation with PNGaseF, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonicacid, and postderivatization cleanup) are performed using 96-well-basedplates. This integrated sample preparation scheme is also compatible withcapillary electrophoresis and MALDI-TOF-MS platforms already inuse in some glycobiology labs and anticipates the higher throughputthat will be offered by the capillary-array-based DNA sequencerscurrently penetrating the market. The described technology should bringhigh-performance glycosylation analysis within reach of each lifesciences lab and thus help expedite the pace of discovery in thefield of glycobiology. [ABSTRACT FROM PUBLISHER]