In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris

Abstract

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.

FIG. 1.

Overview of yeast and mammalian N-linked glycosylation and the strategy used for humanization of P. pastoris glycosylation. In the nonglycoengineered ER Man₉GlcNAc₂ (M9) is trimmed to Man₈GlcNAc₂ (M8) by removal of one α-1,2-mannose residue, after which the glycoproteins are transferred to the cis-Golgi apparatus. In the nonengineered yeast Golgi apparatus N-glycans are further elongated by transfer of one α-1,6-mannose residue by the initiating α-1,6-mannosyltransferase (Och1p). These N-glycans can be further enlarged by several mannosyl- and phosphomannosyltransferases to obtain hypermannose structures. In the mammalian Golgi apparatus, the Man₈GlcNAc₂ structure is shortened to Man₅GlcNAc₂ before addition of a GlcNAc residue by GnTI. Subsequently, two mannose residues are removed by mannosidase II (ManII). The resulting GlcNAcMan₃GlcNAc₂ structure is further modified by several glycosyltransferases, resulting in a complex-type N-glycan. Alternatively, GlcNAcMan₅GlcNAc₂ (GnM5) can be elongated by β-1,4-galactosyltransferase (GalT) to produce a hybrid structure. The lower left panel shows the strategy used and the objectives for glycoengineering of P. pastoris. The enzymes introduced are indicated by solid ellipses. An HDEL-tagged α-1,2-mannosidase is targeted to the ER, trimming all α-1,2-mannose residues. This mannosidase is recognized by the HDEL receptor in the Golgi apparatus and is transported back to the ER by COP I-coated vesicles. Inactivation of OCH1 prevents elongation of the oligosaccharides to hypermannose structures, as the presence of an initiating α-1,6-mannose residue is a prerequisite for further elongation. Further modification occurs by overexpression of Golgi apparatus-localized GnTI and β-1,4-galactosyltransferase. This localization is accomplished by fusion of the catalytic parts of the two enzymes to the sequence that is responsible for targeting of the α-1,2-mannosyltransferase (Kre2p) to the Golgi apparatus of S. cerevisiae. The hybrid structure that is finally obtained is enclosed in a box.

FIG. 2.

OCH1 inactivation vector. Upon digestion of pGlycoSwitchM8 with BstBI and transformation in P. pastoris, the construct integrates at the OCH1 locus. This results in a short OCH1 fragment that does not translate to a functional OCH1 gene and a promotorless fragment that cannot be translated because of the absence of a promoter and the presence of two in-frame nonsense codons.

FIG. 3.

Plasmids used for glycan engineering of P. pastoris.

FIG. 4.

DSA-FACE analysis of N-glycans from different glycan-engineered P. pastoris strains. Panel 1 shows the results for a maltodextrose reference. Panels 2 through 9 show the results for N-glycans, as follows: panel 2, wild-type strain GS115 (the main peak is Man₉GlcNAc₂ [M9]); panel 3, strain with och1 inactivated (the main peak is Man₈GlcNAc₂ [M8]); panel 4, strain with och1 inactivated expressing mannosidase-HDEL (the main peak is Man₅GlcNAc₂ [M5]); panel 5, strain with och1 inactivated expressing mannosidase-HDEL and Kre-GnTI (the main peak is GlcNAcMan₅GlcNAc₂ [GnM5]); panel 6, same as panel 5 but the glycans were treated with β-N-acetylhexosaminidase (the GlcNAcMan₅GlcNAc₂ peak shifted to the Man₅GlcNAc₂ position, indicating that terminal GlcNAc was present); panels 7 and 8, strain with och1 inactivated expressing mannosidase-HDEL, Kre2-GnTI, and Kre2-β-1,4-galactosyltransferase (the additional peak is GalGlcNAcMan₅GlcNAc₂ [GalGnM5]); when the culture was treated with β-galactosidase, the small peak disappeared, as shown in panel 8); panel 9, reference glycans from bovine RNase B (Man_5-9GlcNAc₂ [M5-M9]). RFU, relative fluorescence units.

FIG. 5.

Evaluation of hyperglycosylation after inactivation of P. pastoris OCH1. (A) Coomassie brilliant blue-stained SDS-PAGE gel containing supernatants of P. pastoris strains secreting T. reesei mannosidase. For the nonengineered strain (WT) a clear smear is visible, whereas this smear is not present for the strain with och1 inactivated [och1 (M8)]. (B) FACE analysis of N-glycans derived from mannosidase secreted by a nonengineered strain (WT) and a strain with och1 inactivated [och1 (M8)]. The bands with greater electrophoretic mobility are the Man8 and Man9 bands and represent core N-glycan structures. The hyperglycosyl structures are slowly migrating sugars. They are not present in the strain with och1 inactivated.