N-glycan modification in Aspergillus species

Abstract

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional alpha-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

FIG. 1.

Whole-cell N-glycan analysis of Aspergillus strains expressing engineered α-1,2-mannosidases. The images on the left compare whole-cell N-glycans from A. nidulans recipient strain YR23.5 (A) with strain YR23.5-BC10 (B) and strain YR23.5-CD28 (C). The images on the right show whole-cell N-glycan spectra obtained from A. niger recipient strain T2 (A), strain T2-BC10 (B), and strain T2-CD28 (C). (D) N-glycan spectra obtained after in vitro PNGase F digestion of A. nidulans (left) or A. niger (right) whole-cell extracts with α-1,2-mannosidase. Preparation and analysis of N-glycans was performed as described in Materials and Methods. Structure assignments in this figure and all subsequent figures are based on MALDI-TOF masses (m/z) of individual PNGase F-released ions sensitive to α-1,2-mannosidase, and the relative intensities of the masses are shown (% intensity).

FIG. 2.

MALDI-TOF spectra of N-glycans derived from purified A. niger glucoamylase of the recipient strain T2 (A), strain T2-BC10 (B), or strain T2-CD28 (C).

FIG. 3.

Whole-cell N-glycan analysis of Aspergillus strains expressing engineered GNT I. (A, B, and C) The images on the left compare whole-cell N-glycans from A. nidulans strain YR23.5-BC10, which served as a recipient for the GNT I constructs (A) (shown in Fig. 1B, left, and repeated here for clarity), with strain YR23.5-BC10-coNA15 (B) and strain YR23.5-BC10-mnnJ-NA (C). The images on the right show whole-cell glycan spectra for A. niger strain T2-BC10, which served as a recipient strain for the GNT I constructs (A), compared to N-glycans obtained from strain T2-BC10-coNA15 (B) and strain T2-BC10-mnnJ-NA (C). (D) Schematic diagram representing the function of GNT I, which generates GlcNAcMan₅GlcNAc₂ structures by the addition of one GlcNAc moiety on Man₅GlcNAc₂. GlcNAc residues are represented by squares and mannose (hexose) residues by circles.

FIG. 4.

N-glycan analysis of purified A. niger glucoamylase from strain T2-BC10, which served as a recipient for the GNT I constructs (A) (shown in Fig. 2B and repeated here for clarity), compared to strains T2-BC10-coNA15 (B) and T2-BC10-mnnJ-NA (C).

FIG. 5.

The function of Alg3p. (A) The yeast ALG3 gene encodes the Dol-P-Man:Man₅GlcNAc₂-PP-Dol mannosyltransferase, which converts Man₅GlcNAc₂-Dol-PP to Man₆GlcNAc₂-Dol-PP. (B) Knockout of ALG3 leads to Man₅GlcNAc₂ structures, which can be trimmed to Man₃GlcNAc₂ by α-1,2-mannosidase.

FIG. 6.

Whole-cell N-glycan analysis of Aspergillus strains lacking AlgC activity. (A and B) Whole-cell N-glycan structures of the A. nidulans strain YR23.5-ΔalgC (A) and A. niger strain T2-ΔalgC (B). (C) N-glycan spectrum obtained after in vitro digestion of A. niger T2-ΔalgC whole-cell extracts with purified α-1,2-mannosidase.