Chapter 2 Review of Literature
2.17. Signal processing and glycosylation in Pichia pastoris
Pichia pastoris expression system has the advantage of producing eukaryotic protein products due to its post-translational modifications. However, many disadvantages are associated with the methodology of producing these humanized proteins. Proteins undergo various post translational modification including protein folding and disulphide bridge formation, processing of certain signal sequence, glycosylation (O- and N- linked), majority of which affect the quality and quantity of the protein produced and hence are also known as the ‗rate-limiting step‘. Inefficient protein production occurs mostly due to alterations in these modifications.
2.17.1. Processing of the secretion signals
Pichia pastoris secretes foreign proteins both intracellularly (Payne et al., 2008) and extracellularly (Damasceno et al., 2007). Based on the desired protein expression type i.e. intracellular expression or extracellular secretion use of specific secretion signals can be applied. If the desired protein is not secreted in the native P. pastoris system then inducing it for secretion using signal sequences can lead to decreased yield due to altered protein owing to altered glycosylation or lack of other essential post translational modification. Advantages of intracellular expression in P. pastoris expression system includes cleavage of the amino-terminal methionine residue which affects the
conformational stability, acetylation and also phosphorylation of the amino acid residues such as serine, threonine and tyrosine (O-linked) or histidine (N-linked) of the expressed protein, which are some of the important post translational modifications required in some proteins. However, many disadvantages are also associated with the intracellular expression of proteins in the P. pastoris system including purification due to low intracellular expression (>1%) with progressive reports in only a few proteins such as GFP (Eiden-Plach et al., 2004) or catalase(Shi et al., 2007).
On the other hand, protein secreted can be used with certain specific signal sequences, mostly based on the native secretion signal of the desired protein. Some of the common signal sequences used is the S. cerevisae derived α-MF, P. pastoris derived endogenous acid phosphatase (PHO1) and yeast invertase (SUC2) derived from S. cerevisae. The α- MF, among these, has been most effectively used in many protein secretion such as human epidermal growth factor(Brake et al., 1984), blood factor XII , antibody single chain Fv fragment, human interleukin-17,etc. This is a yeast pheromone consisting of 13- amino acid residues, initially synthesized as 89-amino acid residue composed of pre- and pro- regions consisting of a signal sequence, pro-segment and the repeats of spacer peptides. The pre- regions is reported to direct nascent protein to the endoplasmic reticulum (ER) while the pro- region is reported to direct the processed protein from ER to Golgi apparatus and are cleaved off by signal peptidases(Massahi and Çalık, 2016). In Pichia pastoris secretion of very low levels of its native protein occurs, thus facilitating protein purification. PHO is disadvantageous because it leaves amino acid (Arg) residue at its N terminal compromising its biological study.
2.17.2. Glycosylation in Pichia pastoris
Unlike mammalian cells, Pichia pastoris can undergo N- and O-glycosylation which is of greater importance, mostly in the drug industry. Although the core glycan structure and assembly of the N-glycosylation in both mammals and yeasts are conserved (Man8GlcNAc2), which starts with the transfer of a lipid-linked oligosaccharide unit, Glc3Man9GlcNAc2, to asparagine at the recognition sequence Asn-X-Ser/Thr (X= any amino acid except proline) of the protein, following which the further pattern differ. The mammalian Golgi apparatus performs a series of trimming and addition reactions that generates oligosaccharides composed of either Man5-6GlcNAc2 (high-mannose type), a mixture of several different sugars (complex type) or a combination of both (hybrid type) while proteins secreted from P. pastoris receive much more carbohydrate (50-150 mannose residues) and vizualized by SDS-PAGE and western blotting to be hyperglycosylated/ hypermannosylated. P. pastoris doesn‘t synthesize hyperglycosylated proteins as prominent as other yeast strains such as S. cerevisae. The average chain length of glycoproteins expressed by P. pastoris is only 8–14 mannose residues, whereas that by S. cerevisae is 40∼150 residues. The heterologous protein of Aspergillus awamori glucoamylase produced in P. pastoris was found to be 20kDa heavier than the native protein. About 10 kDa of this weight could be attributed to N-glycosylation, meaning that the rest could be attributed to O-linked glycosides, probably consisting of 20–30 mannose residues, thus concluding that the extent of glycosylation of proteins by P. pastoris was substantially less than that by S. cerevisae. Also, P. pastoris does not undergo addition of α 1, 3-terminal mannose to oligosaccharides unlike S. cerevisae which relieves the antigenic activity compared to proteins produced in S. cerevisae to an extent(Macauley-Patrick et al., 2005). However, addition of outer chain oligosaccharides is still a limitation in the expression of proteins, especially therapeutic proteins as it may
lead to decreased half-life of the protein and also trigger allergic reactions when injected intravenously and thus cleared rapidly by the liver without any proper activity(Ashwell and Harford, 1982). Reports of decreased thermo-stability of various proteins have been observed due to hyper-glycosylation by the P. pastoris expression system. Alkalophilic Bacillus alpha-amylase (ABA) produced in P. pastoris was glycosylated at seven of the nine sites for potential N-glycosylation, as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments and was found to have reduced thermal stability. O-linked glycosylation also differs as the oligosaccharides are mainly composed of mannose residues in contrast to mammals where they are composed of a variety of sugar molecules such as sialic acid, N- acetyl glucosamine and galactose(Bretthauer and Castellino, 1999). Absence of sialic acid also led to rapid clearance of the proteins from the bloodstream of a mammal. O-linked glycosylation were also different in yeasts and mammals in their manner of linkage i.e. unlike in eukaryotic system it doesn‘t add the O-linked oligosaccharide to the hydroxyl groups of preferred amino acids such as serine and threonine. Variations were also observed in the frequency and specificity of glycosylation. Proteins such as human IGF-1 have been reported to be O-linked glycosylated (~15% of the total protein produced) in the P.
pastoris expression system while the protein is not glycosylated at all in its native host.
The mechanisms and specificity of O-glycosylation in P. pastoris is very less exploited(Bretthauer and Castellino, 1999). Reports on a very few heterologous proteins such as Aspergillus awamori glucoamylase catalytic domain, human single-chain urokinase-type plasminogen activator, recombinant human plasminogen, etc. reveal the presence of α 1,2-mannans containing dimeric, trimeric, tetrameric, and pentameric oligosaccharides and absence of α 1,3 linkages were detected. Production of recombinant human antithrombin III from P. pastoris showed that the O-glycosylation of the protein
occurred near the reactive site and resulted in the recombinant protein having half the inhibitory activity against thrombin when compared with the native anti-thrombin III(Bretthauer, 2003).
Due to the above problems, various glyco-engineered strains have been attempted.
Recombinant strains of P. pastoris containing an integrated cDNA for either secreted haemagglutinin or influenza neuraminidase were further transformed with a full-length cDNA for α-1, 2-mannosidase from Trichoderma reesei along with the ER retention signal (HDEL) for the S. cerevisae MNS1 protein (specific ER-processing α-1, 2- mannosidase) which were reported to further reduce the hyper-glycosylation and also proper localization signal for α-1, 2-mannosidase in the ER. Other modifications have also been developed such as deletion of OCH1 gene encoding the mannosyl transferase responsible for hyper-glycosylation. Co-expression of the trans-sialidase with the Trichoderma reesei mannosidase, under the control of the GAP promoter, gave a secreted trans-sialidase that contained predominantly Man5GlcNAc2. Expression of the trans-sialidase under the control of the AOX1 promoter in the absence of co-expressed mannosidase were reported to be void of terminal 1,6-linked mannose residues typically found in P. pastoris(Choi et al., 2003). Also engineered strains with an inhibitor of the major ER located protein-O-mannosyl transferases (PMTs) with reduced O- glycosylation were developed since mannosyl phosphorylation has been observed in several recombinant proteins which altered its structural and functional properties.
Fusion FC-protein have also been reported to successfully be expressed in glyco- engineered Pichia pastoris strain(Jacobs et al., 2008; Laukens et al., 2015).