Figure 1.
N-glycosylation and engineering thereof in yeast. (A) N-glycosylation in wild type yeast and (B) The approach used to engineer the yeast specific pathway.
A: Standard N-glycosylation pathway in the ER. The early steps in N-glycosylation start with the synthesis of a dolichol-linked Man5GlcNAc2 glycan precursor that flips to the ER lumen, where it is further elongated with mannoses starting with the activity of Alg3p mannosyltransferase. The resulting dolichol-linked Man9GlcNAc2 precursor is then also glucosylated starting with the activity of Alg6p glucosyltransferase. When complete, the Glc3Man9GlcNAc2 glycan is transferred en bloc to the nascent polypeptide chain. These glycans are then subjected to a protein folding quality control process involving de-glucosylation by glucosidases I and II (GI, GII) and re-glucosylation glucosyltransferase. B: The engineering strategies used to obtain a Y. lipolytica strain that produces glycoproteins homogeneously modified with the trimannosyl core N-glycan (Man3GlcNAc2). First, ALG3 was knocked out (1), then Alg6p was overexpressed (2), then GII was overexpressed (3), and finally α-1,2-mannosidase was overexpressed (4). Conforming to the representation proposed by the Consortium for Functional Glycomics Nomenclature Committee, the green and blue spheres represent mannose (Man) and glucose (Glc), respectively, and blue squares represent N-acetylglucosamine residues (GlcNAc). C: Man3GlcNAc2-glycans can be further modified to any complex-type N-glycan structure using a combination of glycosyl-transferases, either in vitro or in vivo.
Table 1.
Y. lipolytica strains used in this study.
Figure 2.
Identification of N-glycans by exoglycosidase digestion and DSA-FACE analysis.
A: Oligomaltose reference. B, N-glycans from RNaseB reference. C–G, N-glycans from different strains: C, MTLY60 wild type strain; D, alg3 knock-out strain; E, The same as D but treated with α-1,2-mannosidase; F, The same as D but treated with JB α-mannosidase; G, The same as D but treated with glucosidase II. The N-glycan structures in the alg3 knock-out strain are consistent with Man5GlcNAc2, GlcMan5GlcNAc2 and Glc2Man5GlcNAc2.
Figure 3.
DSA-FACE analysis of engineered Y. lipolytica strains.
A, oligomaltose reference. B–K, N-glycans derived from different sources: B, bovine RNaseB reference; C, MTLY60 wild type strain; D, alg3 knock-out strain; E, alg3 mutant strain overexpressing Alg6p. F–J, the alg3 mutant strain overexpressing Alg6p engineered with: F, Y. lipolytica GIIα; G, Y. lipolytica GIIα HDEL-tagged; H, both α and β subunits of Y. lipolytica GII; I, the HDEL-tagged A. niger GIIα; J, both α and β subunits of A. niger GII. K, The latter strain engineered with an HDEL-tagged T. reesei α-1,2-mannosidase. This fully engineered strain produces glycoproteins with more than 85% trimannosyl core N-glycans.
Figure 4.
SDS-PAGE evaluation of underoccupancy of N-glycan sites in lipase 2 after inactivation of alg3.
1, Wild-type strain (WT, MTLY60). 3, The same as lane 1 but overexpressing lipase2. 5, The alg3 knock-out strain overexpressing lipase 2 and Alg6p. 7, The alg3 knock-out strain overexpressing lipase2. Lanes 2, 4, 6 and 8, the same as 1, 3, 5, and 7, respectively, but treated with PNGaseF. A hyperglycosylation smear is observed when lipase2 is overexpressed in the WT strain. For the alg3 mutant strain expressing lipase2, two distinct bands are visible, which is consistent with site underoccupancy largely compensated for by Alg6p overexpression. Lane 9: PNGaseF preparation used for the digestions shown in Lane 2, 4, 6 and 8.
Figure 5.
T. brucei GII and mutanase tested as engineering approach.
(A) The dual N-glycosylation system in T. brucei. Both Man9GlcNAc2 and Man5GlcNAc2 can be transferred to proteins. Next, these proteins are reglucosylated and deglucosylated in the folding cycle by glucosyltransferase and GII, respectively. (B) DSA-FACE analysis of reference N-glycans and N-glycans derived from strains engineered with T. brucei GII or treated with mutanase. A, Oligomaltose reference. B, N-glycans from RNaseB reference. C, N-glycans from the alg3 mutant strain overexpressing Alg6p. D-F, N-glycan from the alg3 mutant strain overexpressing Alg6p and engineered in different ways: D, engineered with T. brucei GII; E, engineered with T. brucei GII with HDEL tag; F, engineered with T. brucei GII with HDEL tag and pre-lip2 signal. G, N-glycans derived from the alg3 mutant strain overexpressing Alg6p treated with mutanase.
Table 2.
Primers used in this study.