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GlycodExpress™ and YAC-Express™: two innovative technologies developed for N-glycosylation Humanization and Production of Therapeutic Recombinant Glycoproteins in Saccharomyces cerevisiae. Christine Bonnet, Christelle Arico, Céline Rigaud, Claire Blandais, Emilie Chanteclaire, Emilie Tassy-Freches and Christophe Javaud. Glycode S.A.S, 6 rue porte Baffat, 19140 Uzerche, France. Corresponding author: [email protected] or [email protected]
Bibliography 1. Jez J., Antes B., Castilho A., Krainer M., Wiederkum S., Grass J., Rüker F., Woisetschläger M. and Steinkellner H. (2012) Significant impact of single N-glycan residues on the biological activity of Fc-based antibody like fragments. J. Biol. Chem. 287(29): 24313-9. 2. Böhm S., Schwab I., Lux A. and Nimmerjahn F. (2012) The role of sialic acid as a modulator of the inflammotory activity of IgG. Semin. Immunopathol. 34(3)443-53. 3. Lam J.S., Mansour M.K., Specht C.A. and Levitz S.M. (2005) A model vaccine exploiting fungal mannosylation to increase antigen immunogenicity. J. Immunol. 175(11): 7496-503. 4. Chiba Y., Suzuki M., Yoshida S., Yoshida A., Ikenaga H., Takeuchi M., Jigami Y. and Ichishimai E. (1998) Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae. J. Biol. Chem. 273(41): 26298. 5. Lehle L., Eiden A., Lehnert K., Haselbeck A. and Kopetzki E. (1995) Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation. FEBS Lett. 370(1-2): 41-5. 6. Kotake T., Hojo S., Tajima N., Matsuoka K., Koyama T. and Tsumurava Y. (2008) A bifunctional enzyme with L-Fucokinase and GDP-L-Fucose Pyrophosphorylase activities salvages free L-Fucose in Arabidopsis. J. Biol. Chem. 283(13): 8125. 7. Coyne M.C., Reinap B., Lee M.M. And Comstock L.E. (2005) Human symbionts use a host-like pathway for surface fucosylation. Science 307: 1778.
Glycode patents WO/2008/095797. Genetically modified yeasts for the production of homogeneous glycoproteins. WO/2012/013823. A Yeast Artificial Chromosome carrying the mammalian glycosylation pathway.
Acknowlegments: We thank Jean-Noël Chaize and Cora Merelli from the Glycode fermentation platform for yeast microfermentation, and Ludivine Perrocheau and Pauline Guéraud from the Glycode biochemistry and purification platform for downstream process of the total secreted proteins.
Background As budding yeast is a GRAS organism, devoid of intellectual property limitation, there is for years an increasing interest on its utilization for industrial purposes. Saccharomyces cerevisiae is a particularly efficient and attractive system for production of recombinant proteins. Indeed, growth in neutral and well defined culture media is no time-consuming and cheaper than in higher eukaryotic systems. Large scale production allows high yields and post-translationally modified proteins can be more easily secreted and purified, than in other systems. However, glycosylation is essential for mammalian glycoproteins stability, targeting and biological activity (1,2) and, as proteins produced in wild type strains of yeast carry glycans radically different from those of the natural one, they can present less activity and changes in their immunogenicity (3). Engineering of Saccharomyces cerevisiae glycosylation is a prerequisite at its use as an efficient production system of therapeutic proteins. First step of this process consists in the deletion of the yeast glycosylation pathway. Heterologous glyco-enzyme active domains fused to yeast localization sequences are introduced in the genome. Finally, availability and accessibility of sugar donors are essential for the glyco-enzymes functionality,
Results We show that OCH1 and MNN1 sequences, glyco-enzymes involved in host N-glycosylation process, have to be deleted to obtain more than 90% of homogeneity of core glycan structures. Depending of mother strain background, other mannosyltransferases (MNN9, MNN4…) may be deleted if they take over at this stage of engineering. The a-1,2-mannosidase screening process illustrates the method to select an optimal fusion between a heterologous glyco-enzyme active domain and a yeast localization sequence. Our work was inspired by Chiba results (4) and we obtained a significant percentage of the Man5GlcNAc2 structure when the glyco-enzyme was ectopically expressed in the yeast (25-50 % depending of the strain). This percentage was increased, even doubled, after genomic integration. Nucleotide-sugar donors are essential for the building of humanized glycan structures. Some of them, as GDP-fucose and CMP-sialic acid are not present in yeast. Glycode has developed innovative methods for their synthesis. The approach concerning GDP-fucose is presented.
Conclusion Glycode has developed two innovative technologies to humanize N-glycosylation in yeast Saccharomyces cerevisiae. In the GlycodExpress™ technology (WO/2008/095797), Saccharomyces cerevisiae has been modified by sequential deletion of mannosyltransferases and introduction of glycosyltransferases expression cassettes into the yeast genome, by homologous recombination. It offers several advantages: strong stability in time, full tracking of genotypic and phenotypic changes and possibility to reengineer the strain in an identical way, in case of spontaneous recombination. For the YAC-Express™ approach (WO/2012/013823), Saccharomyces cerevisiae has been engineered by introduction of a YAC containing a cluster of glycosyltransferases expression cassettes in a yeast strain deleted for various mannosyltransferases. Among the numerous advantages of this technology, the absence of direct modification in the yeast chromosomes, the short delay for engineering a new strain and the fact that YAC technology can be extended to a wide variety of strains can be noted.
GlycodExpress™ technology YAC-Express™ technology
Presented at the 2012 IUBMB-FEBS congress, Sevilla, Spain.
Mannosyltransferases deletion
Heterogeneity of N-glycans in wild type strains
N-glycans released from secreted glycoproteins with PNGase F were analysed by mass spectrometry (MALDI-TOF) at Proteodynamics, Clermont-ferrand, France. H1, Y1 and B1 : wild type Saccharomyces cerevisiae strains.
Glycans analysis of various wild type Saccharomyces cerevisiae strains reveal hypermanno-sylation but also an obvious heterogeneity in polymannose structures.
OCH1 gene deletion
N-glycans released from secreted glycoproteins with PNGase F were analysed by mass spectrometry (MALDI-TOF) by Proteodynamics. H2, Y2, B2 and D2: Doch1 Saccharomyces cerevisiae strains.
As earlier describe (5), OCH1 deletion impaires the elongation of the glycan outer chain, however a significant heterogeneity remains concerning polymannose structures (Man8-11 GlcNAc2).
MNN1 gene deletion in Doch1 strains
Permethylated N-glycans released from secreted glycoproteins with PNGase F were analysed by mass spectrometry (MALDI-TOF) by Proteodynamics. H3 (upper spectrum), Y3 and D3 (lower spectrum): Doch1
Dmnn1 Saccharomyces cerevisiae strains.
M8
M9
M10
H3
M8
M9
M10
D3 Phosphomannoses
Homogeneous polymannose glycan structures (up to 90% of Man8-9 GlcNAc2) are obtained after deletion of MNN1 in Doch1 strains. Further engineering may be needed (MNN9 or MNN4 deletion) according to additional structures detected.
This poster will be available on www.glycode.fr
Glyco-enzymes screening Screening strategy
Glyco-enzyme active domains from various origines Localisation sequences (cytosol / ER / Golgi apparatus : cis, trans…)
Surexpression in Saccharomyces cerevisiae strains
Glycan analysis on total secreted proteins
a-1,2-mannosidase selection M5
M6
M7
M8
M9
M5
M6 M7
M8
M9
Mns1-CE: Caenorhabditis elegans a-1,2-mannosidase fused with Saccharomyces cerevisiae MNS1 localisation sequence. Mns1-AS: Aspergillus saitoi a-1,2-mannosidase fused with Saccharomyces cerevisiae MNS1 localisation sequence. AS-HDEL: : Aspergillus saitoi a-1,2-mannosidase fused with the HDEL retention motif.
Mass spectrometry analysis (MALDI-TOF) of permethylated N-glycans released from secreted glycoproteins with PNGase F. (Proteodynamics). Upper spectrum: Y3 + AS-HDEL Lower spectrum: H3 + AS-HDEL Glycoenzymes are expressed by a multicopy plasmid, under constitutive promoter TEF (high strength)
Among the different construction tested, best results were obtained with the A. saitoi a-1,2-mannosidase fused, in C-term, to the HDEL retention signal.
Targeted genomic integration
M6
M7 M8
M9
M5
Mass spectrometry analysis (MALDI-TOF) of permethylated N-glycans released from secreted glycoproteins with PNGase F. analysis carried out by Proteodynamics. Y4: a-1,2-mannosidase is constitutively expressed (under TEF promoter), by an expression cassette integrated in 1 copy on the yeast genome.
Genomic integration enhances selected a-1,2-mannosidase activity and the percentage of Man5GlcNAc2 increases to 50%.
Y3+AS-HDEL
H3+AS-HDEL
GDP-fucose synthesis
L-fucose Fucose-1- phosphate GDP-fucose
Fucokinase activity Pyrophosphorylase activity
Mn2+
ATP ADP GTP PPi
Pyruvate kinase
Pyruvate Phosphoenol Pyruvate
Pyrophosphorylase
Pi + Pi
MESG
methylpurine Phosphorylase
Ribose-1-phosphate
NADH
NAD+
Lactate deshydrogenase
L-lactate
Monitoring NAD+ apparition (OD at 340nm)
Monitoring MESG disparition (OD at 360nm)
Activities tests
GDP-fucose synthesis pathways
GDP-mannose GDP-D-mannose GDP-fucose GDP-mannose-
4,6-deshydratase GDP-4-keto-6-deoxy-D-Mannose/4-reductase
Principal described mammalian pathway: 2 enzymes
L-fucose Fucose -1-P GDP-fucose Fucose Kinase GDP-fucose
pyrophosphorylase
Salvage described mammalian pathway: 2 enzymes
L-fucose Fucose -1-P GDP-fucose
Glycode innovating pathway: only 1 bifunctional enzyme
fucokinase/L-fucose-1-P-ganylyltransferase
Enzymes from two distinct organisms, A. thaliana (6) and B. fragilis (7) have been tested for activities by surexpression in Y1 wild type strain
Fucokinase activity
-0,2
-0,15
-0,1
-0,05
0
0,05
0,1
5 10 15 20
DO
D 3
60
nm
Time (mn)
Pyrophosphorylase activity
■ Wild type strain
■ Strain expressing the A. thaliana enzyme
■ Strain expressing the B. fragilis enzyme
The enzymatic activities of Fkp (bacterial enzyme) and FKGp (plant enzyme) was assayed in vitro, on fresh yeast extract. Each mesure was done in triplicate, in a final volume of 200mL . OD evolution was followed on a microplate reader (TECAN).
Tested enzymes are functional, for both activities, in the yeast Saccharomyces cerevisiae. However, activities developed by Bacteroides fragilis Fkp is significantly higher: this enzyme will be chosen for futher engineering.
-0,1
-0,05
0
0,05
0,1
0,15
5 10 15 20DO
D 3
40
nm
Time (mn)