Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures.

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man5GlcNAc2 glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man3-5GlcNAc2 species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man3GlcNAc2 glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex6-7HexNAc2 structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man3-5GlcNAc2 species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.

SnapShot: N-Glycosylation Processing Pathways across Kingdoms.

Post-translational modification of proteins with carbohydrates shapes their localization and function. This SnapShot presents the core pathways from different organisms that install these complex and highly variable structures.