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WO2013169009A1 - Plante pour la production de n-glycane riche en mannose, et procédé de production de n-glycane riche en mannose à l'aide de celle-ci - Google Patents

Plante pour la production de n-glycane riche en mannose, et procédé de production de n-glycane riche en mannose à l'aide de celle-ci Download PDF

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WO2013169009A1
WO2013169009A1 PCT/KR2013/004043 KR2013004043W WO2013169009A1 WO 2013169009 A1 WO2013169009 A1 WO 2013169009A1 KR 2013004043 W KR2013004043 W KR 2013004043W WO 2013169009 A1 WO2013169009 A1 WO 2013169009A1
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mannose
plant
glycan
gnt1
gnti
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이균오
이상열
유재용
고기성
손보화
드위 파나타 와유인드라
릭노하르모코
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경상대학교 산학협력단
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a plant for producing a high-mannose type N-glycan and a method for producing a high-mannose type N-glycan using the same.
  • the endoplasmic reticulum and the golgi apparatus are organelles that play an important role in the processing of various proteins that are transported to the cell membrane, cell walls, vacuoles, lysosomes or secreted out of the cell.
  • the messenger RNA (mRNA) which encodes proteins in the secretory pathway including membrane proteins, begins the synthesis of polypeptides, including the signal sequence that moves to the endoplasmic reticulum when combined with ribosomes, and the newly produced polypeptide removes the signal sequence from the endoplasmic reticulum membrane.
  • N-glycosylation has a significant effect on the biochemical properties of proteins such as protein folding, assembly, migration and activity (Ruiz-Canada et al. , 2009).
  • intrinsic glycosylation of proteins plays a very important role in determining biological properties and activity.
  • Protein glycosylation is divided into two categories, N-linked and O-linked modifications. These two forms differ in the amino acid to which the glycan moiety is attached. That is, O-bonds are attached to Serine or Threonine (Thr) residues, while N-bonds are attached to Asparagine (Asn) residues.
  • Glycans in either N-linked or O-linked form have their own unique structural characteristics and are known to significantly affect the function of glycoproteins, including biopharmaceuticals. In particular, N-glycan processing is more frequently found in glycoproteins, and research on the function and disease of proteins related to N-glycosylation is being actively conducted.
  • GnTI N-acetylglucosaminyltransferase I
  • GnTI corresponds to an enzyme essential to make GlcNacMan 5 GlcNac 2 form N-glycan by adding ⁇ 1,2-linked GlcNAc to the basic N-glycan of Man 5 GlcNac 2 form.
  • ⁇ 1,2-linked GlcNAc by GnTI is an essential process that must be preceded for N-glycan maturation, which requires that ⁇ 1,3-fucose, ⁇ 1,2 Hybrid and complex type N-containing plant-specific monosaccharides such as xylose, ⁇ 1,3-galactose and ⁇ 1,4-fucose Glycan glycoproteins can be made. Therefore, eukaryotic cells lacking the gene of the enzyme do not produce the maturation of N-glycans, so mixed and complex N-glycan glycoproteins are not produced, but instead, high-mannose-type N-glycan glycoproteins are produced. It is known.
  • Lysosomal accumulation disease is a large class of rare genetic diseases that can cause serious clinical signs. About 50 lysosomal accumulation diseases are known due to the lack of lysosomal enzymes, carriers or proteins associated with lysosomal biosynthesis or lysosomal function. The overall incidence of lysosomal accumulation disease is about 1 in about 7000 newborns.
  • a representative disease of lysosomal accumulation disease is Gaucher disease. Goche disease is caused by a deficiency of an enzyme called glucocerebrosidase bound to a lysosome membrane that catalyzes the hydrolysis of glucocerebroside (glucosylceramide, GlcCer), a glycosphingolipid composed of glucose and ceramide.
  • Glucocerebrosidase deficiency is caused by mutations in the human glucocerebrosidase gene located at the q21-q31 region of chromosome 1, and the characteristic storage cells called goche cells are found in the spleen and liver of patients with Goche disease. Found in bone marrow, etc.
  • Related clinical symptoms include hepatosplenomegaly, anemia, thrombocytopenia and skeletal deteriora-tion.
  • the sequence of the gene encoding human glucocerebrosidase was first determined in 1985.
  • the protein consists of 497 amino acids derived from 536-mer pro-peptides.
  • Human glucocerebrosidase has five N-glycosylated amino acid consensus sequences (Asn-X-Ser / Thr). Four of these sites are normally glycosylated and the high-mannose type and complex N-glycans are processed. Glycosylation of the first site is essential for the production of proteins with activity.
  • Human glucocerebrosidase isolated from the placenta contains 7% carbohydrate sites, 20% of which are in high-mannose form.
  • Biochemical studies and site-specific mutation studies on glucocerebrosidase are the initial maps of regions and residues that are important for folding, activator interaction, and active site location. map).
  • enzyme replacement therapy has been developed as a treatment for these disorders, which has recently been treated with Cerezyme TM , an enzyme produced by genetic recombination technology. The most successful results have been reported in patients with Shefian disease.
  • Protein drugs such as beta-glucocerebrosidase, which are used to treat lysosomal accumulation disorders, require the delivery of proteins through mannose receptors present on the surface of macrophages.
  • Glycosidases such as neuraminidase, galactosidase and hexosaminidase are produced to have high-mannose-type N-glycans instead of glycans. .
  • this production method has a long and complicated production process has a lot of influence on the stability and production cost of protein therapeutics.
  • a glycoprotein drug having a mannose terminal and a protein therapeutic agent for lysosomal accumulation disease including beta-glucocerebrosidase, which is used as a drug for the treatment of Goche disease in order to solve the problems of the present technical field as described above.
  • UDP-GlcNAc ⁇ 3-D-mannoside ⁇ 1,2-N-acetylglucosaminyltransferase I
  • UDP-GlcNAc alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I; GnTI
  • the purpose of this study is to identify callus derived from rice and its seeds whose gene expression and N-acetylglucosaminyltransferase I lost function and to use them to produce protein medicines with high-mannose type N-glycans.
  • Another object of the present invention is a method for producing a high-mannose type N- glycan protein using a gnt1 mutant plant or its callus, which has lost the function of N-acetylglucosaminyltransferase I (GnTI). To provide.
  • Another object of the present invention is to provide a method for screening and analyzing high-mannose-type N-glycans, which have lost the function of N-acetylglucosaminyltransferase I (GnTI). will be.
  • the present invention provides a gnt1 mutant plant producing a high-mannose type N-glycan, which has lost the function of N-acetylglucosaminyltransferase I (GnTI). .
  • the present invention also provides the production of high-mannose type N- glycan proteins using gnt1 mutant plants, seeds or callus which have lost the function of N-acetylglucosaminyltransferase I (GnTI). Provide a method.
  • the present invention also provides a method for screening and analyzing high-mannose-type N-glycans, which have lost the function of N-acetylglucosaminyltransferase I (GnTI).
  • the gnt1 mutant plant according to the present invention loses the function of UDP-GlcNAc: ⁇ 3-D-mannoside ⁇ 1,2-N-acetylglucosaminyltransferase I (GnTI), which is a mixed and complex type when using the plant. It is possible to inhibit the production of N-glycan glycoprotein and instead to produce high-mannose N-glycan glycoprotein. Therefore, the protein having a high-mannose N-glycan according to the present invention can be used in glycoprotein drugs having a mannose terminal according to the protein therapeutic agent for lysosomal accumulation disease, and thus can be usefully used in the field of protein medicine.
  • FIG. 1 is a diagram showing the complementation of the Arabidopsis by expression of rice GnTI (complemention). Formation of complex and mixed N-glycans using anti-horseradish peroxidase (anti-HRP), anti-fucose and anti-xylose antibodies Comparison was made by immunoblot and the amount of high-mannose type N-glycans was determined by lectin blot analysis using concanavalin A.
  • Figure 2 is a diagram confirming the mutant inserting T-DNA into gnt1 in rice.
  • 2 (a) shows the genomic structure of rice GnTI and T-DNA insertion sites.
  • 2 (b) shows the deletion of 44 nucleotides in gnt1 by T-DNA insertion.
  • 2 (c) shows genotyping of heterozygous (He) gnt1 , homozygous (Ho) gnt1 and WT using the indicated primer pairs.
  • 2 (d) shows RT-PCR analysis of HE, Ho and WT.
  • 2 (e) shows DNA gel blot analysis of HE, Ho and WT.
  • Figure 3 shows the expression analysis of GnTI and adjacent genes in gnt1 and WT. Expression of genes encoding cation release family protein (Os0 2 g58580), GnTI protein (Os0 2 g58590), hypothetical protein (Os0 2 g58600) and protein kinase (Os0 2 g58610) in WT and GnTI, respectively. .
  • FIG. 4 shows an immunoblot affinity blot of the entire leaf protein obtained from gnt1 and WT.
  • the blots were labeled with anti-horseradish (HRP), anti-fucose, anti-xylose antibodies, and affinity blot analysis was performed using concanavalin A; con A) / peroxidase system was used.
  • HRP anti-horseradish
  • confucose anti-fucose
  • anti-xylose antibodies anti-xylose antibodies
  • N-acetylglucosamine square
  • fucose triangle
  • xylose asterisk
  • mannose black circle
  • galactose residues empty circle
  • FIG. 6 is a diagram showing the results of MALTI-TOF mass spectrometry of PNGase A-releasing permethylated N-glycans in gnt1 callus.
  • N-acetylglucosamine square
  • fucose triangle
  • xylose asterisk
  • mannose black circle
  • galactose residues empty circle
  • Figure 7 shows the HPLC profile of fluorescently labeled N-glycans in gnt1 and WT.
  • N-acetylglucosamine square
  • fucose triangle
  • xylose asterisk
  • mannose black circle
  • galactose residues empty circle
  • the present invention provides a gnt1 mutant plant that produces a high-mannose type N- glycan protein that completely inhibits the expression of the gnt1 gene and thus loses the function of N-acetylglucosaminyltransferase I (GnTI). to provide.
  • GnTI N-acetylglucosaminyltransferase I
  • the gnt1 mutant plant according to the present invention is characterized by producing a high-mannose type N-glycan protein by losing the function of N-acetylglucosaminyltransferase I (GnTI).
  • the plant is not limited thereto, but may be food crops, vegetable crops, special crops, fruit trees, flowers and feed crops, preferably rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, sorghum, baby poles. , Cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut, rapeseed, apple tree, Pear, jujube, peach, peach, grape, citrus, persimmon, plum, apricot, banana, rose, gladiolus, gerbera, carnation, chrysanthemum, lily, tulip, lygras, redclover, orchardgrass, alfalfa, tolfscue and It may be selected from the group consisting of perennial grass.
  • the experiment can be effectively lacking the expression in plants, including the baby
  • Glycoylation is known to have a significant effect on protein activity, folding, assembly, migration, stability, solubility, sensitivity to proteases, half-life and antigenicity.
  • Glycan processing is one of the most common post-translational modificaiton. Protein glycosylation is divided into two categories, N-bonds and O-bonds. These two forms differ in the amino acids to which the glycan site is attached on the protein.
  • high-mannose-type N-glycan refers to a glycan having one or more exposed mannose residues.
  • a plant in which the function of N-acetylglucosaminyltransferase I (GnTI) is completely lost can be prepared.
  • the method of losing the function of the GnTI is known in the art, that is, gene deletion, gene insertion, T-DNA insertion, homologous recombination or transposon tagging.
  • Etc. may be used, and preferably, the gene of UDP-GlcNAc: alpha 3-D-mannoside ⁇ 1,2-N-acetylglucosaminyltransferase I (GnTI) in a transformation population of a plant into which T-DNA is inserted.
  • the method of selecting the plant ( gnt1 ) which lost the function of N-acetylglucosaminyltransferase I by lack of expression was used.
  • the loss of GnTI function may be achieved by removing part or all of the nucleotide sequence represented by SEQ ID NO: 1.
  • the nucleotide sequence may be removed by introducing T-DNA into the plant, but the present invention is not limited thereto, and the process of preparing a dysfunctional plant by inserting T-DNA into the GnTI gene is a general method known in the art. It can be made by.
  • a method of inserting the T-DNA gene into the plant may be used.
  • Preferred examples of recombinant vectors are Ti-plasmid vectors capable of transferring the T-DNA region which is part of itself when present in a suitable host such as Agrobacterium tumefaciens to the chromosomal DNA of the plant cell.
  • a particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • the Ti-plasmid vector may preferably comprise one or more selectable markers.
  • the marker is typically a nucleic acid sequence having properties that can be selected by biochemical methods, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes include, but are not limited to.
  • the host cell may use any host cell known in the art, and is preferably Agrobacterium tumefaciens .
  • the host cell is preferably a plant cell.
  • the method of carrying the vector of the present invention into a host cell is performed by the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973) when the host cell is a prokaryotic cell). ), Hanahan method (Hanahan, D., J. Mol.
  • one T-DNA is inserted into the GnTI gene so that expression of the GnTI gene is reduced.
  • analyze the isolation rate of herbicide or antibiotic resistance in the selection medium or use similar genes in T-DNA as probes as probes to probe similar sequences in the genomic DNA of the transgenic plant. It is preferable to select a line of rice with one T-DNA inserted into the transformant through Southern blot analysis, which hybridizes and finds a gene having the same.
  • the left- and right-border regions of the T-DNA are amplified by PCR and T It is preferable to analyze the position of the T-DNA inserted into the rice GnTI gene and the removed gene sequence by flanking sequence analysis technique which clones the vector and analyzes the sequence. At this time, T-DNA is inserted into the exon region of the GnTI gene or a part of the exon is removed in order to secure an individual lacking the expression of the GnTI gene, thereby inducing a frame shift mutation or a nonsense mutation. It is more desirable to screen the isolated individuals.
  • Plants in which the T-DNA is inserted into the GnTI gene preferably include analyzing the loss of function of N-acetylglucosaminyltransferase I.
  • the activity of N-acetylglucosaminyltransferase I can be analyzed by any method known in the art, preferably ⁇ 1,3-fucose, ⁇ 1,2-xylose ( Immunoblotting and Concanavalin A to detect plant specific monosaccharides and complex N-glycans using 1: 10000 dilution, Agrisera) and horseradish peroxidase (HRP) (1: 20000 dilution, Sigma) antibodies, respectively.
  • HRP horseradish peroxidase
  • Con A can be analyzed by affinity blotting to detect high-mannose type N-glycans, or the entire N-glycans can be analyzed by peptide: N-glycanase A. After digestion with (peptide: N-glycanase A: PNGase A), the entire N-glycan profile can be analyzed by MALDI-TOF mass spectrometry.
  • the present invention also provides a method for producing a high-mannose type N-glycan protein using a plant lacking GnTI gene expression or its callus.
  • the method for producing the glycoprotein includes expressing a high-mannose-type N-glycan protein in a plant and purifying the protein, but not limited thereto, and include a method known in the art.
  • a method for purifying the N-glycan protein there are methods such as salting, dialysis, chromatography and electrophoresis.
  • the method of centrifugally separating salts having different solubility by changing salt concentration or pH is generally used.
  • Molecular sieve filtration chromatography that separates ion-chromatography or cross-linked molecules between molecules based on the size and shape of the molecule using a difference in electrostatic interaction with a carrier may be used in all commonly used methods.
  • Affinity chromatography using specific intermolecular interactions, ultracentrifugation method that separates biomaterials in a density gradient of a solution or sugar using a difference in sedimentation rate for centrifugal force may be possible, but is not limited thereto. It may include all conventional methods known in the art.
  • Step (a) is a method for applying a PCR reaction to the mRNA of the plant dml primer from which the gnt 1 gene is removed, thereby preparing cDNA of the plant.
  • reverse transcriptase is performed as a previous step using reverse transcriptase and oligo (dT) primer.
  • quantitative real time polymerase chain reaction qRT-PCR is an experimental method based on polymerase chain reaction. Real-time polymerase chain reaction simultaneously amplifies and measures target DNA molecules. For one or more specific sequences in a DNA sample, real-time polymerase chain reaction allows detection and quantitation. The measurement can count the absolute number of copies or the relative amount.
  • the method of the RT-PCR reaction and q-RT-PCR reaction is not limited to the following examples, and may include all known in the art.
  • Step (b) is a step of selecting plants that produce high-mannose-type N-glycans, and whether the production of high-mannose-type N-glycans can be confirmed from the plants using an immunoblot and an affinity blot. have.
  • the immunoblot and affinity blot include peroxides (POD), alkaline phosphatides, ⁇ -galactosidase, eurease, catalase, glucose oxidase, lactic acid dehydrogenase, amylase or biotin-avidin complex, and the like.
  • POD peroxides
  • alkaline phosphatides ⁇ -galactosidase
  • eurease catalase
  • glucose oxidase lactic acid dehydrogenase
  • amylase or biotin-avidin complex
  • Enzymes may be used, and in the case of fluorescence immunoassays, fluorescein isothiocyanate, tetramethylhodamine isocyanate, substituted rhodamine isothionate, dichlororiazin isothiocyanate, Alexa or Alexa Fluorescent materials such as fluoro (Alexa Fluoro) or fluorophores may be used, and radioisotopes such as tritium, iodine, phosphorus and sulfur may be used for radioimmunoassay, and luciferase method and luminol peroxy are used for luminescence immunoassay. Days POD method or the like can be used with luminescent materials such as dioxetane d.
  • the antibody may be combined with the labeling substance as needed, such as when using the avidin-biotin method, the streptoavidin-biotin method, and the like.
  • a method such as glutaraldehyde method, maleimide method, pyridyl disulfide method, periodic acid method, etc. may be used for enzyme immunoassay, and chloramine T method and Bolton-hunter for radioimmunoassay. Methods such as law may be used.
  • immunological measurement methods include, but are not limited to, immunoprecipitation, immunoblotting, western blotting, immunostaining, immunodiffusion, and the like. It may include all methods (immunoblotting).
  • the step (c) is to identify plants that produce high-mannose-type N-glycans by purifying and analyzing N-glycans.
  • the method for purifying N-glycan protein includes protein solubility, charge, and molecular weight. , Purifying according to adsorption or affinity for other biomolecules, and protein purification in a biological system can be used the same method as a general protein.
  • Methods for purifying and analyzing the N-glycan protein can be confirmed by known methods.
  • Known methods include all methods known in the art, such as chromatography, electrophoresis and mass spectrometry.
  • HPLC high performance liquid chromatography
  • MALDI-TOF matrix-assisted laser desorption / ionization
  • PCR polymerase chain reaction
  • the stress sensitive phenotype of cgl1 is known to be caused by incomplete N- glycan maturation of glycoproteins (Kang et al., 2008).
  • T-N- cgl1 the article Mau maturation of stress sensitivity to check whether the result in the restoration of phenotype, the roots of the WT, and T-cgl1 rice GnTI- expressed cgl1-T on the growth medium containing the salt recovered from (C3 and C7 line) was analyzed. The results are shown in FIG. 1 (E).
  • Mutants with T-DNA inserted into GnTI were obtained from a collection of T-DNA mutants produced at Genoplante (http://www.genoplante.com/). Genomic DNA was extracted from the leaves and callus of rice using phenol-chloroform. PCR reactions using a combination of specific primers [P2 + LB] and [P1 + P2] were used to confirm the insertion site and homozygosity of T-DNA. T-DNA insertion sites were recovered by PCR using specific primers [P2 + LB] and [P1 + P2] and cloned into pGEM-T vector (Promega, http://www.promega.com/). Analysis was by sequencing. The primers used in the present invention are shown in Table 1 below.
  • P3 (5'-CCGAAAACCAATGCACCAGTCAAC-3 ') and P4 (5'-TGTATGTTCTGCAGACTTCCGGGC-3') were used as forward and reverse primer sets, respectively.
  • Actin 1 primer was used as a control of RNA content.
  • PCR reaction conditions are as follows; Once, 2 minutes at 94 ° C. (denatured); 28 times, 15 seconds at 94 ° C. (denatured), 30 seconds at 60 ° C. (restored), 1 minute at 70 ° C. (extension); And once, 5 minutes at 70 ° C.
  • the qRT-PCR was performed using the CFX96 ® Detection System (Bio-Rad , http://www.bio-rad.com/) and SsoFast ® Eva-Green ® Supermix ( Bio-Rad) RT-PCR mixture. OsAct1 was amplified and used as an internal positive control.
  • the BLAST search of the TIGR rice genome annotation database with the rice cDNA (AJ457976) sequence as a query shows that a GnTI gene consisting of 18 exons exists as a single copy at the Os0 2 g58590 locus. It was shown.
  • Table 2 shows the isolation ratio of heterozygous gnt1 mutants obtained from progeny in a medium containing hygromycin.
  • Table 2 shows the total number of seedlings tested and the number of seedlings showing resistance or susceptibility to hygromycin. The c 2 test was used to assess the number of copies of T-DNA insertions and the significance level was 0.05.
  • Genomic DNA of 10 kinds of microorganisms obtained from the release bonding gnt1 mutants or homozygous wild-type rice callus gnt1 mutant was digested with the BamHI and EcoRI.
  • the DNA fragments were separated by electrophoresis on 0.8% agarose gel and nylon membrane (Hypobond-N +, GE Healthcare, http: //www.gelifesciences) by capillary transfer using a salt transfer protocol. .com).
  • DNA was immobilized on the membrane by UV cross-linking of 1200 ⁇ J cm ⁇ 2 and washed for 5 minutes at 2 ⁇ SSC.
  • HPT-F and HPT-R primer pairs were then used to produce T-DNA specific probes by PCR using genomic DNA from the gnt1 mutant as a template.
  • Membranes were hybridized with 32 P-labeled T-DNA specific probes and washed under moderate conditions. The blot was visualized by autoradiography by exposure to x-ray film in a cassette with a sensitizing screen at 70 ° C. for 24 to 72 hours.
  • sequencing PCR fragments prepared using gene specific (P1 and P2) primers and T-DNA (LB and RB) primers revealed that T-DNA was inserted into GnTI and 6 exons ( 44 nucleotides (nt), each containing 42 nt) and six introns (2 nt), were deleted and replaced by T-DNA.
  • T-DNA insertion at six exon / intron junctions leads to abnormal splicing of GnTI and introduction of immature termination codons, which ultimately leads to post-transcriptional mRNA degradation. Let's do it.
  • Plant (rice) protein extracts were performed as described above (Strasser et al., 2004) using three- week- old gnt1 mutants and wild type seedlings for immunoblot and affinity blot analysis.
  • Total protein extracts of 10 microorganisms were dissolved in 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Hybond-ECL, Amersham).
  • the membranes were subjected to ⁇ 1,3-fucose, ⁇ 1,2-xylose (Agrisera) or HRP (Sigma, http: //www.sigmaaldrich). labeled with an antibody against .com /). Concanavalin A was used to measure high-mannose N-glycans by immunoblotting.
  • anti-horseradish, anti-fucose, anti-xylose antibodies responded only to proteins derived from wild type, but not to gnt1 mutants. This showed that the formation of pocimanose and complex N-glycans comprising core ⁇ 1,3-fucose and ⁇ 1,2-xylose residues lost function in the gnt1 mutant.
  • concanavalin A showed strong reaction with proteins derived from the gnt1 mutant, but weakly with proteins derived from the wild type, indicating that the amount of high-mannose N-glycans in the gnt1 mutant It was confirmed that the increase.
  • Proteins for N-glycan purification were extracted from callus as described above (Bakker et al., 2006). The protein was digested with trypsin and the immobilized N-glycans were released by PNGase A (Roche, http://www.roche.com/). After digestion, the sample was passed through a C18 Sep-Pak cartridge and then lyophilized. The carbohydrate fractions were dissolved in dimethylsulfoxide and then permethylated as described above (Anumula and Taylor, 1992).
  • glycoproteins extracted from embryo-derived callus were used for matrix-assisted laser-desorption time-of-flight mass spectrometry analysis. The results are shown in FIGS. 5, 6 and Table 3.
  • N-glycans obtained from wild-type (WT) callus showed various complex N-glycans and Lewis (Le a ) epitopes including ⁇ 1,2-xylose, ⁇ 1,3-fucose ( epitope) was expressed in the WT callus.
  • N-glycans derived from the gnt1 mutant callus consist mostly of high-mannose N-glycans (Man 4-9 GlcNAc 2 ) (76.7%) with Man 5 GlcNAc 2 in major form, Mannose and complex N-glycans were not found.
  • Table 3 shows the results of MALDI-TOF MS analysis of N-glycans obtained from WT and gnt1 callus.
  • the figures represent the percentage of total peak area. [ND, not detected; Man, mannose; Xyl, xylose; GlcNAc, N-acetylglucosamine; Fuc, fucose; Gal, galactose]. Quantitative analysis of N-glycans obtained from WT and gnt1 was performed with MALDI-TOF MS.
  • N-glycans obtained from WT and gnt1 were performed by MALDI-TOF mass spectrometry using N-glycans generated as derivatives by permethylation prior to analysis (Wada et al., 2007).
  • the spectra shown were generated from various spectra of 100 laser shots.
  • the integrated peak area of the entire integrated isotope population was determined by relative quantitative analysis.
  • HPLC profiling was performed using an Asahipak NH2P-50 column (Shodex, Kunststoff, Germany) at a flow rate of 1.0 ml / min.
  • 70% Solvent A 200 mM; 1: 9 acetic acid / triethylamine (pH 7.3): acetonitrile ratio
  • 30% Solvent B 200 mM; 9: 1 acetic acid / triethylamine (pH 7.3): The column was equilibrated with the solution using a solution containing a ratio of acetonitrile). After injecting the sample, the proportion of solvent B increased linearly to 45% for 45 minutes.
  • PA-oligosaccharides were measured by fluorescence using a model 275 fluorescence detector (Waters, Milford, Mass.) (Excitation and emission wavelengths of 320 and 400 nm). Passed fractions were collected every minute using a fraction collector for peak identification by mass spectrometry (Microflex TOF, Bruker Daltonik GmbH, Bremen, Germany).
  • Table 4 above shows the HPLC analysis of N-glycans obtained from WT and gnt1 callus.
  • the figures represent the percentage of total peak area. [ND, no detected; Man, mannose; Xyl, xylose; GlcNAc, N-acetylglucosamine; Fuc, fucose; Gal, galactose].
  • Quantitative analysis of N-glycans obtained from WT and gnt1 was performed by HPLC using N-glycans generated as derivatives by permethylation prior to analysis (Wada et al., 2007). Peak areas were determined by relative quantitative analysis.
  • the present inventors have found that the lack of GnTI activity in rice completely inhibits N-glycan maturation in Golgi, and the defect of N-glycan maturation has a Man5GlcNAc 2 form, high-mannose N- It was confirmed that the accumulation of glycans leads to major living organisms.

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Abstract

La présente invention concerne une plante pour la production de N-glycane riche en mannose, et un procédé de production de N-glycane riche en mannose à l'aide de celle-ci. La plante mutante pour gnt1 de la présente invention perd la fonction de UDP-GlcNAc:α3-D-mannoside β1,2-N-acétylglucosaminyltransférase I (GnTI), et par conséquent il est possible d'inhiber la production de glycoprotéines N-glycanes combinées et complexes et de produire des glycoprotéines N-glycanes riches en mannose à la place, lorsqu'on utilise la plante. Par conséquent, la protéine comprenant le N-glycane riche en mannose de la présente invention peut être utilisée dans des applications médicales de glycoprotéines et similaires ayant une extrémité terminale mannose correspondant à une protéine thérapeutique pour des maladies de stockage lysosomal, et par conséquent peut être utile dans le domaine des fournitures médicales protéiques.
PCT/KR2013/004043 2012-05-08 2013-05-08 Plante pour la production de n-glycane riche en mannose, et procédé de production de n-glycane riche en mannose à l'aide de celle-ci WO2013169009A1 (fr)

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WO2020190031A1 (fr) * 2019-03-18 2020-09-24 (주)지플러스 생명과학 Plante transgénique à expression supprimée de cgl1 et cgl2 et procédé de production d'une protéine cible l'utilisant

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US20110008837A1 (en) * 2007-06-15 2011-01-13 Marc-Andre D-Aoust Modifying glycoprotein production in plans
WO2011119115A1 (fr) * 2010-03-25 2011-09-29 Agency For Science, Technology And Research Méthode de production de protéines recombinantes comprenant des n-glycanes à terminaisons mannose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110008837A1 (en) * 2007-06-15 2011-01-13 Marc-Andre D-Aoust Modifying glycoprotein production in plans
WO2011119115A1 (fr) * 2010-03-25 2011-09-29 Agency For Science, Technology And Research Méthode de production de protéines recombinantes comprenant des n-glycanes à terminaisons mannose

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FANATA, WAHYU INDRA DUWI ET AL.: "N-glycan maturation is crucial for cytokinin- mediated development and cellulose synthesis in Oryza sativa", THE PLANT JOURNAL, vol. 73, no. 6, March 2013 (2013-03-01), pages 966 - 979 *
SARIBAS, A. SAMI ET AL.: "Refolding of human beta-1-2 GlcNAc transferase (GnTl) and the role of its unpaired Cys 121", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 362, no. 2, 13 August 2007 (2007-08-13), pages 381 - 386 *
VON SCHAEWEN, A. ET AL.: "Isolation of a mutant Arabidopsis plant that lacks N-Acetyl Glucosaminyl Transferase I and is unable to synthesize Golgi-Modified Complex N-Linked Glycans", PLANT PHYSIOLOGY, vol. 102, August 1993 (1993-08-01), pages 1109 - 1118 *

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