Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides

Miura, Masami; Hirose, Masaaki; Miwa, Taeko; Kuwae, Shinobu; Ohi, Hideyuki

doi:10.1016/j.gene.2003.09.023

Cited by 21 publications

(13 citation statements)

References 33 publications

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“…the sequon Asn-Xaa-Ser/Thr, which has been found to be necessary for N -linked glycosylation, but is not always sufficient [22]. The PNO1 (phosphomannosylation of N -linked oligosaccharides) gene was cloned and was found to promote N -linked glycosylation only to the core oligosaccharides and not to the outer sugar chain, unlike the MNN4 gene of S. cerevisiae [74]. This would explain why proteins expressed in P. pastoris have shorter glycosylation chain lengths than those expressed in S. cerevisiae.…”

Section: O-and N-linked Glycosylationmentioning

confidence: 99%

“…One group of researchers disrupted the PNO1 gene of P. pastoris, resulting in a reduction of N -liked glycosylation of a recombinant human antithrombin. They reported that the glycosylation had fallen from around 20% in the wild-type strain to less than 1% in the engineered strain [74], showing that suppression of N -linked glycosylation is possible.…”

Section: O-and N-linked Glycosylationmentioning

confidence: 99%

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Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,085

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Section: O-and N-linked Glycosylationmentioning

confidence: 99%

Section: O-and N-linked Glycosylationmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,085

633

View full text Add to dashboard Cite

show abstract

“…Two Mnn4-like genes exist in S. cerevisiae (19), four exist in P. pastoris (4,27) and Ogataea minuta (1), and eight exist in Candida albicans (17). However, in Y. lipolytica, only a single gene (YALI0D24101g) was predicted to encode a type II membrane protein showing homology with ScMnn4p.…”

Section: Discussionmentioning

confidence: 99%

Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

Park

Song

Cheon

et al. 2011

Appl Environ Microbiol

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Yeast species are important hosts for the production of therapeutic secretory proteins owing to their ability to secrete and glycosylate proteins, their rapid growth to high cell density, and the ease with which they can be genetically manipulated. However, the glycosylation pathway of yeast cells is known to be different from that of mammalian cells, and yeast glycans could induce immunological responses in humans (5). In the traditional yeast Saccharomyces cerevisiae, the typical characteristics of the Asn (N)-linked glycan include hypermannosylation (Man 50-150 GlcNAc 2 ), mannosylphosphorylation in the core and outer regions, and termination with the ␣1,3-linked mannose residue. These yeast-specific sugar moieties could prove problematic in the production of therapeutic glycoproteins; hypermannosylation can impair protein activity, and mannosylphosphate residues and terminal ␣1,3-linked mannoses may elicit an antigenic response (14).Several nonconventional yeast species, including the methylotrophic species Pichia pastoris and Hansenula polymorpha, have emerged as alternative systems for the production of recombinant therapeutic proteins. Advantages over S. cerevisiae include reduced hypermannosylation GlcNAc 2 ), no terminal ␣1,3-linked mannose of N-linked oligosaccharides, and efficient heterologous protein secretion (18,20,21,22). As another alternative expression system for the production of human-derived therapeutic glycoproteins, the dimorphic yeast Yarrowia lipolytica has recently drawn attention since this yeast naturally secretes several enzymes, including proteases, lipases, esterases, and RNases, at elevated levels and its posttranslational modification ability is similar to that of mammalian systems (3,26,29).Recently, we reported that the N-linked glycans of Y. lipolytica are composed of neutral and acidic sugars lacking a terminal ␣1,3-linked mannose (38). The neutral sugars of Nlinked glycans are high-mannose oligosaccharides, principally Man 7-12 GlcNAc 2 , and the acidic sugars of N-linked glycans are composed of monomannosylphosphorylated Man 7-9 GlcNAc 2 sugars. In the case of S. cerevisiae, at least four mannosylphosphorylation loci have been detected in the core and outer chain regions of N-linked glycans, and the mannosylphosphorylation of N-linked glycans in the Golgi apparatus requires both S. cerevisiae Mnn4p (ScMnn4p) and ScMnn6p (19). Deletion of ScMNN4 or ScMNN6 induced a significant reduction in alcian blue intensity, which reflects the presence of negatively

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“…A recent patent application by GlycoFi Inc. (WO 2005/106010 A2) describes the cloning of four -1,2-mannosyltransferase genes from P. pastoris, named AMR1-4 (for alpha-mannosidase resistant). Disruption of AMR2 in an och1 pno1 mnn4B background (with PNO1 [200] and MNN4B (patent application number WO2005065019 A2) probably being two mannosylphosphate transferases) resulted in complete conversion of all alpha-mannosidase resistant structures (comprising up to 20% of the total N-glycan pool in the och1 pno1 mnn4B strain) to Man 5 GlcNAc 2 following -1,2-mannosidase treatment.…”

Section: Towards Homogeneous Complex-type N-glycansmentioning

confidence: 99%

N-glycosylation Engineering of Biopharmaceutical Expression Systems

Jacobs

Callewaert²

2009

CMM

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N-glycosylation, the enzymatic coupling of oligosaccharides to specific asparagine residues of nascent polypeptide chains, is one of the most widespread post-translational modifications. Following transfer of an N-glycan precursor in the ER, this structure is further modified by a number of glycosidases and glyco-syltransferases in the ER and the Golgi complex. The processing reactions occurring in the ER are highly conserved between lower and higher eukaryotes. In contrast, the reactions that take place in the Golgi complex are species- and cell type-specific. Due to its non-template driven nature, glycoproteins typically occur as a mixture of glycoforms. Since N-glycans influence circulation half-life, tissue distribution, and biological activity each glycoform has its own pharmacokinetic, pharmacodynamic and efficacy profile. Moreover, modification of glycoproteins with non-human oligosaccharides can result in undesired immunogenicity. Therefore, engineering of the N-glycosylation pathway of most currently used heterologous protein expression systems (bacteria, mammalian cells, insect cells, yeasts and plants) is actively pursued by several academic and industrial laboratories. These research efforts are in the first place directed at humanizing the N-glycosylation pathway and eliminating immunogenic glycotopes. Moreover, one wants to establish new structure-function relationships of different glycoforms, which helps to decreasing the complexity of the N-glycan repertoire towards one defined N-glycan structure. In this review, we discuss the most important recent milestones in the glycoengineering field.

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Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides

Cited by 21 publications

References 33 publications

Heterologous protein production using the Pichia pastoris expression system

Heterologous protein production using the Pichia pastoris expression system

Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

N-glycosylation Engineering of Biopharmaceutical Expression Systems

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