Abstract
BackgroundTherapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches.ResultsIn this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry.ConclusionThis strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.
Highlights
Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals
Expression of Endo‐T on the surface of Pichia pastoris Endo-T is the first fungal member of glycoside hydrolase family 18 with endo-beta-N-acetylglucosaminidase or endoglycosidase (ENGase)-type activity secreted from Hypocrea jecorina (Trichoderma reesei) [44]
In the GlycoDelete glycoengineering strategy, Endo-T has been successfully expressed in the Golgi of mammalian cells and plants to produce recombinant protein with homogenous N-glycan structures [17, 18], or to enhance integral membrane protein with homogenous N-glycans between the two N-acetylglucosamine (GlcNAc) expression in P. pastoris [45]
Summary
Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. Wang and collaborators used an EndoA mutant (N171A) to glycosylate IgG1-Fc region [21, 23, 28, 29], and further used the mutants of Endo-S (D233A and D233Q) or Endo-S2 (D184M and D184Q) for fulllength antibody glycosylation remodeling with three major types (complex, high-mannose, and hybrid type) of N-glycans for modulating IgG effector function [14, 22, 30] This chemoenzymatic glycosylation method utilizing ENGases provides an efficient way to introduce complex N-glycans onto polypeptides, which was valuable for glycoprotein drug production [13, 31]. N-Glycosyltransferase AaNGT and ApNGTQ469A were reported to transfer GlcN and produce N-GlcNAc glycans by coupling with GlmA [16, 33]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
