The Research Of Glycosylation Engineering In Pichia Pastoris And Characterization Of WFDC2

The methylotrophic yeast Pichia pastoris is used extensively as the host cell for large-scale production of secreted recombinant proteins. Many proteins of pharmaceutical inportance are N-glycosylated, and therefore require an expression host that yields N-liked oligosaccharides that are structurally and functionally identical to the human counterpart. But 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 30-50 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. In first part of my study, we describe engineering of the P. pastoris N-glycosylation pathway according to mammalian cell expression system to produce nonhyperglycosylated hybrid glycans-GlcNAcMan5GlcNAc2-Asn.a -1,6-mannosyltransferase (OCH1) is the first key enzyme in Pichia glycosylation pathway that different from mammalian cells. It can add a-1,6-mannose at the end of MangGlcNAc2 to form Man9GlcNAc2 oligosaccharide sugar, then this oligosaccharide as a substrate continue added a series of mannose, and ultimately the formation of high mannose type. So a-1,6-mannosyltransferase (OCH1) is a key enzyme leads to generation of high mannose type, and OCH1 gene must be knockout, as Pichia sugar the initial step based engineered, blocking the Pichia high mannose glycosylation. First, the OCH1 gene of P. pastoris were knocked out by a two-step homologous recombination method. In which the MazF-ZeoR cassette was inserted between the OCH1(a-1,6-mannosyltransferase) homologous arms, Escherichia coli toxin gene mazF and zeocin resistance gene acted as counter-selectable and active-selectable markers respectively. This novel method allows repeated use of the zeocin for multiple modifications and will be a useful tool for P. pastoris studies. The glycan analysis shows that 85% of the recombinant WFDC2 protein glycans is Man8GlcNAc2, the hyperglycosylation is blocked sucessfully.Man8GlcNAc2 is then turned to Man5GlcNAc2 structure by a-1,2-mannosidase I which located on Golgi, Man5GlcNAc2 is the intermediate type formed hybrid type sugar. So exogenous a-1,2-mannosidase I need to be introduced to synthesis this sugar chain structure in Pichia. In this study, a-1,2-mannosidase I (Mns I) from T. reesei is introduced by arginine auxotrophic screening. The S.cerevisise a-mating factor was used to replace the signal peptide of a-1,2-mannosidase I gene from T. reesei, after introduction of this DNA fragment fused with endoplasmic reticulum(ER) retention signal sequence(HDEL),68% the mammalian cells-like mannose-type (Man5GlcNAc2) sugar chain was observed by HPLC-ESI-MS of N-linked glycans from WFDC2 protein produced in KMochl stain, showing the Mns I from T. reesei can cleave N-glycans from Man8GlcNAc2 to Man5GlcNAc2.After Man5GlcNAc2 transffered into the intermediate Golgi, which is formed into hybrid type sugar chain GlcNAcMan5GlcNAc2 by the β-1,2-N-acetylglucosamine transferase I (GnT I). Then GlcNAcMan5GlcNAc2 enters the outer Golgi, further formed into complex sugar chains. In order to target the human β-1,2-N-acetylglucosaminetransferase I (GnT I) to the Golgi apparatus, the GnT I N-terminal portion was replaced by the S.cerevisise MNN9 Golgi-retainng location signal gene. The N-linked glycans analyzed result showed the human GnT I can transfer a β-N-acetylglucosaminyl to Man5GlcNAc2, the result showed 91% of Man5GlcNAc2 was converted to GlcNAcMan5GlcNAc2 glycan.In the second part, the glycoengineered P. pastoris were used to express WFDC2 protein and study the effects of glycosylation on WFDC2’s biochemical characterization. WFDC2 is a 124 amino acid long polypeptide with one glycosylation site, which is a member of whey acidic protein four-disulfide core (WFDC2) family with not clear function. A WFDC protein has conserved WAP domain of 50 amino acids with eight conserved cystine residue, which is supposed to have anti-protease and anti-bacterical activities. Here, we study the activities of WFDC2 expressed in wild type P. pastoris and glycoengineered P. pastoris with human embryonic kidney (HEK) 293F cells derived WFDC2 protein as a control, which attaches a hypermannosylation/hybrid glycans and complex glycans.The results showed that WFDC2 protein was found bound to both Gram-negative and Gram-positive bacteria, especially with Staphylococcus aureus, but it demonstrated subtle inhibitory activity towards the growth. Moreover, WFDC2 protein exhibited proteinase inhibitory activity towards trypsin, elastase, MMP9 and the secretory proteinases from Bacillus subtilis, MMP9 was significantly reduced by WFDC2. To investigate the effect of carbohydrate structure on WFDC2 protein, WFDC2 protein with high-mannose/hyper-glycan showed lower biological activity compared with complex-glycan modified WFDC2, the KMoGnT I-WFDC2 showed similar trypsin inhibitory with 293F-WFDC2, which demonstrate that the activities altered by the different carbohydrate structures.In conclusion, we developed a Pichia N-glycan engineering stain, which allows GlcNAcMan5GlcNAc2 glycosylation. This strain made for further N-glycosylation engineering. Furthermore, the biological activity of WFDC2 and the effects of glycosylation on WFDC2’s biochemical characterization in vitro was first reported, which may facilitate further research examining the structure of WFDC2 and its role in the physiolgical and pathology process.