Recombinant Expression And Characterization Of O-Mannose Or N-GlcNAc Modified Heterologous Proteins In Pichia Pastoris

Glycosylation is the most common post-translational modification in eukaryotic cells.The structures of glycans play important roles in the function of glycoproteins.The methylotrophic yeast,Pichia pastoris,is a single-cell eukaryote microorganism with simple post-translational modifications.This organism is capable of performing both N-and O-linked glycosylation.In P.pastoris,the N-glycan is high mannose type with an average of 8-14 mannose residues.P.pastoris cells also typically assemble O-linked glycans,which are linear chains of four to five ?-linked mannose residues and may be capped with ?-or phospho-mannose,on hydroxy-amino acids such as serine and threonine.With the development of glycoengineered P.pastoris expression system,more and more glycoproteins were expressed using P,pastoris as a host.In the first part of our work,we expressed and characterized the recombinantly expressed O-glycoprotein Ala-Pro-rich antigen(Apa)from Mycobacterium tuberculosis to compare the substrate specificities of O-mannosyltransferases in P.pastoris and M.tuberculosis.In the second part,we constructed a platform to produce GlcNAc-linked glycoproteins by expressing Beta-acetyl endoglucosidases(ENGases)in ER,Golgi or on cell surface,which provide the scaffold for further in vitro syntheses of functional N-linked glycans on P.pastoris expressed proteins.O-mannosylation,which is widely present in bacteria,fungi,and animals,performs extremely important functions,such as interactions between cell and extracellular matrix,as well as pathogen adhesion and invasion.In M.tuberculosis,Apa is one of the main immune antigen and has the potential as a vaccine and diagnostic reagent for tuberculosis.Apa extracted from M.tuberculosis is unambiguously O-glycosylated on four threonine residues(Thr10,Thr18,Thr27 and Thr277),each with 1-3 mannose units via ?-1,2 glycosidic linkages.The O-mannose glycosylation of Apa is essential for its biological function in the process of colonization and invasion of host cells by M.tuberculosis and deglycosylated or nonglycosylated Apa proteins failed to stimulate T lymphocyte response.Here,the gene encoding the mature Apa was amplified from M.tuberculosis H37Rv genomic DNA and expressed in P.pastoris GS115.The glycosylation of recombinant Apa(rApa)was analyzed by lectin blot and matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS).The results showed that the rApa was secreted into the fermentation broth by P.pastoris with a yield of approximately 0.6 g/L.The western blot of PNGase F-treated rApa showed that the Apa produced by P pastoris was modified not only with N-linked glycans but also with O-mannose units.The addition of O-glycans was further confirmed by MS-assisted peptide map analyses.In addition to the four known M.tuberculosis-derived glycosites,some other Thr/Ser residues could also be glycosylated.Furthermore,P.pastoris can recognize the known glycosites of M.tuberculosis Apa.Both the glycosites and the length of the glycans of recombinant Apa are similar to those in the native protein.Thus,P pastoris has the potential for preparation of vaccines or diagnostic reagents against TB.In addition,P.pastoris could be used as a platform to investigate the substrate specificities of different PMT isoforms which catalyze the addition of mannose motifs onto certain T/S residues.N-linked glycosylation is one of the most common post-translational modifications involved in protein folding,pharmacokinetic stability and function.Currently,there are more than 300 biotherapeutics on the market,including monoclonal antibodies,hormones,and growth factors,most of which are N-linked glycoproteins.Due to the differences in N-glycosylation patterns between diverse species,most therapeutic glycoproteins produced by recombinant expression systems carrying non-human N-glycan structures which may cause undesired immune response,even allergic reaction.ENGases are a class of enzymes that acts on the linkage between two GlcNAc residues in the N-glycosylation core structure,producing glycopeptide or glycoprotein with only one GlcNAc motif.Besides hydrolysis,some ENGases of GH85 family possess transglycosylation activity,i.e.,the ability to transfer the structure-defined and uniform sugar chain to the GlcNAc residue of glycopeptide or glycoprotein.Due to its extensive research and biological effects,ENGase has great potential for the production of pharmaceutical proteins.In our work,the glycosidase Endo-T derived from Hypocrea jecorina was localized and expressed in ER or Golgi and cell surface of P.pastoris,constructing recombinant strains to produce a single GlcNAc residue-linked glycoprotein.To assess the efficiency of N-glycan remodeling,human GalNAc-T1 and human IgG Fc domain having different number of N-glycans,were stably expressed in recombinant strains and extracted from the culture media.The results showed that the locations of Endo-T in P.pastoris affect its glycosidase activities.Endo-T exhibits the higher deglycosylated activity in Golgi apparatus,of which the pH value closes to the optimum pH of Endo-T function(pH 6.0).This glycol-engineered platform might be possible to apply in producing important therapeutic mammalian N-glycoproteins.