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WO1992009694A2 - CLONAGE DE UDP-N-ACETYLGLUCOSAMINE:α-3-D-MANNOSIDE β-1,2-N-ACETYLGLUCOSAMINYLTRANSFERASE I - Google Patents

CLONAGE DE UDP-N-ACETYLGLUCOSAMINE:α-3-D-MANNOSIDE β-1,2-N-ACETYLGLUCOSAMINYLTRANSFERASE I Download PDF

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WO1992009694A2
WO1992009694A2 PCT/CA1991/000417 CA9100417W WO9209694A2 WO 1992009694 A2 WO1992009694 A2 WO 1992009694A2 CA 9100417 W CA9100417 W CA 9100417W WO 9209694 A2 WO9209694 A2 WO 9209694A2
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WO1992009694A3 (fr
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Harry Schachter
Mohan Sarkar
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Hsc Research And Development Limited Partnership
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention relates to DNA sequences for the human and rabbit enzymes which control the conversion of high mannose to hybrid and complex N-glycans, UDP-N- acetylglucosamine: ⁇ -3-D-mannoside jS-l,2-N- acetylglucosaminyltransferase I (GnT I) , plasmids containing such DNA sequences, transformed cells containing such plasmids, and a method for converting high mannose glycoproteins to branched N-glycan glycoproteins.
  • N-glycans share the common core structure Man ⁇ l-6(Man ⁇ l-3)Man/?l-4GlcNAc?l-4GlcNAc/3-Asn.
  • Complex N-glycans have "antennae" or branches attached to this core.
  • the antennae are initiated by the action of at least five Golgi-localized membrane-bound GlcNAc-transferases designated GnT I, II, IV, V and VI (Schachter et al (1989) Methods Enzvmol. , vol. 179, 351-396) and may be further elongated by the addition of D-galactose, L-fucose and sialic acid residues.
  • N-glycans may be "bisected" by a GlcNAc residue attached in ?l-4 linkage to the ⁇ -1inked Man of the core due to the action of GlcNAc-transferase III (GnT III) .
  • R is GlcNAc/31-4(+/-F uc ⁇ l ⁇ 6 )
  • GlcNAc ⁇ Asn- ⁇ and Asn-X may be an Asn residue which is part of the amino acid sequence of a protein.
  • the enzyme is specific for the Man ⁇ l-3Man?l-4GlcNAc-arm of the core.
  • the presence of a 02-linked GlcNAc residue at the non-reducing terminus of this arm is essential for subsequent action of several enzymes in the processing pathway (Schachter et al (1983) Can. J. Biochem. Cell Biol. , vol. 61, 1049-1066; Schachter et al (1985) "Glycosyltransferases involved in the biosynthesis of protein-bound oligosaccharides of the asparagine-N-acetyl-D-glucosamine and serine(threonine)-N-acetyl-D-galactosamine types", in: A.N. Martonosi, ed.
  • GnT II has been reported in hen oviduct, Chinese hamster ovary cells, baby hamster kidney cells, bovine colostrum, pig trachea and mammalian liver (Schachter et al (1983) Can. J. Biochem. Cell Biol. , vol.
  • the cloning of DNA encoding proteins and the expression of such cloned DNA to produce the proteins has become commercially important.
  • a primitive host such as a bacteria (e.g., E. coli) , a yeast, or a fungus.
  • a primitive host such as a bacteria (e.g., E. coli) , a yeast, or a fungus.
  • such primitive hosts may not normally possess the enzymes required for the post-translation modification of proteins which occurs in the cells from which the DNA originated.
  • many primitive hosts possess the necessary enzymes to effect the post-translation modification of a protein to a high mannose derivative such host do not contain the enzyme required to convert the high mannose derivative to a hybrid and branched glycan, GnT I.
  • Yeast and vertebrate cells use the same Glc 3 Man 9 GlcNAc.
  • lipid-linked precursor for cotranslational glycosylation of asparagine residues both recognize the same Asn-X-ser/Thr sequences, and both remove the three glucose residues soon
  • a mammalian glycoprotein expressed in yeast may contain the same carbohydrate chains as the native protein until after it leaves the endoplasmic reticulum. After entry into the Golgi, however, the later steps in oligosaccharide processing are very different in yeast (see Kukuruzinska et al, Ann. Rev. Biochem. , vol. 56, p.915, 1987) and vertebrates, (see Hubbard and Ivatt Ann. Rev. Biochem. r vol. 50, p.555, 1981; Kornfeld and Kornfeld Ann. Rev. Biochem. , vol. 54, p.631, 1985).
  • oligosaccharides contain two GlcNAc residues and from 9 to 50 or more mannose residues.
  • mammalian oligosaccharides never have more than nine mannose residues and most commonly contain GlcNAc, galactose, and sialic acid attached to a Man 3 GlcNAc 2 core.
  • heterologous expression in yeast of a mammalian glycoprotein intended for therapeutic use can present a number of potential glycosylation-related problems.
  • carbohydrate chains may be highly antigenic; in addition, they are recognized by Man/GlcNAc-specific receptors on cells of the mammalian reticuloendothelial system, resulting in rapid clearance of the glycoprotein from the circulation.
  • Figure 1 illustrates the amino acid sequence data for the eight peptides isolated from rabbit liver GnT I and nucleotide sequences of the six synthetic oligonucleotides prepared on the basis of the peptide sequences.
  • the single letter code is used for amino acid sequence data; upper case letters indicate firm assignments and lower case letters indicate tentative assignments.
  • the underlined sections of the peptide sequences indicate the regions used for the design of oligonucleotide probes.
  • Probes 2, 3 and 6 were based on peptides 2, 3 and 6, respectively; S indicates "sense” and A indicates "antisense" directions;
  • Figure 2 illustrates a schematic representation of GnT I clones.
  • PCR product product obtained by PCR amplification of rabbit liver cDNA; re 1600, 1.6 kb GnT I cDNA clone; rc2500, 3.0 kb GnT I cDNA clone.
  • the shaded boxes represent the coding region.
  • the 3.0 kb cDNA was reduced to 2.5 kb by a 0.5 kb deletion at the 5'-end;
  • SUBSTITUTE SHEET Figure 3 illustrates the results of an agarose gel electrophoresis (1% agarose) of the products of the polymerase chain reaction (PCR) using rabbit liver cDNA as template and the following combinations of oligonucleotides as primers; 2S-3A; 2S-6A; 3S-2A; 3S-6A; 6S-2A; 6S-3A ( Figure 1) .
  • Conditions of PCR are given in the Methods section.
  • the gel was stained with ethidium bromide (0.5 ⁇ g/ l) .
  • Primer-dependent products were obtained with combinations 2S-6A (0.50 kb) and 3S-6A (0.45 kb) .
  • the arrow designates the 0.5 kb DNA marker; the remaining standards are at 1.0 kb, 1.6 kb, 2.0 kb and at 1.0 kb intervals thereafter;
  • Figure 4 illustrates the nucleotide sequence (lower case) of the 2.5 kb GnT I cDNA clone.
  • the amino acid sequence in the coding region is shown in upper case letters.
  • the positions of the eight peptide sequences obtained from proteolytic digests of GnT I ( Figure 1) are underlined with a single solid line; the regions of these peptide sequences used for oligonucleotide probe synthesis ( Figure 1) are additionally underlined with a discontinuous line.
  • the putative transmembrane segment (bases 62-136) is underlined with a double line.
  • the consensus polyadenylation signal AATAAA at position 2435 is underlined.
  • Figure 5 illustrates an autoradiogram of an SDS- polyacrylamide gel electrophoresis experiment showing in vitro transcription and translation of the rabbit cDNA.
  • mRNA was generated from the 2.5 kb GnT I cDNA and was used as the template for in vitro translation using rabbit reticulocyte lysate and L-[ 35 S]-methionine (see Methods for details).
  • Lane C no plasmid in the incubation; lane 12, pGEM-7z containing the 2.5 kb GnT I cDNA with an insert between bases 56 and 57 which interrupts the reading frame; lane 16, pGEM-7z containing the 2.5 kb GnT I cDNA (pGEM-7z-rcgntl) ;
  • Figure 6 illustrates the nucleotide sequence for human geno ic DNA encoding for GnT I
  • Figure 7 illustrates the amino acid sequence for human GnT I
  • Figure 8 illustrates both the nucleotide sequence for human genomic DNA encoding for GnT I and the amino acid sequence of human GnT I.
  • one aspect of the present invention relates to isolated DNA sequences which encode rabbit GnT I.
  • DNA sequences encode a protein having the sequence (starting from the N-terminal) of formula I shown below:
  • the present invention relates to DNA sequences which encode human GnT I .
  • DNA sequences encode a protein having the sequence (starting from the N-terminus ) of formula II shown below:
  • Exemplary of the DNA sequences encoding rabbit GnT I is the sequence (starting from the 5'-terminus) of formula III, shown below: atg ctg aag aag cag tct get ggg ctt gtg ctg tgg ggt get ate etc ttt gtg gcc tgg aat gee ctg ctg etc etc ttc ttc tgg aca cgt cca gtg cct age agg ctg ccg tea gac aat get etc gat gat gac cct gcc age etc ace cgt gag gtg ate cgc tta get cag gat gcc gag gta gag ttg gaa cgt cag egg gga ctg ttg cag cag att agg gag cae cat get ctt
  • the DNA sequence of formula III corresponds to the coding region of rabbit cDNA encoding GnT I.
  • Another example of a DNA sequence encoding rabbit GnT I is a larger section of cDNA encoding rabbit GnT I, which has the formula
  • Exemplary of the DNA sequences encoding human GnT I is the sequence (starting at the 5 '-terminus) of formula V, shown below: atgctgaa gaagcagtct gcagggcttg tgctgtgggg cgctatcctc tttgtggcct 961 ggaatgccct gctgctcctc ttctgga cgcgcccagc acctggcagg ccaccctcag 1021 tcagcgctct cgatggcgac cccgccagcc tcacccggga agtgattcgc ctggcccaag 1081 acgccgaggt ggagctggag cgcaggcgtgctgca gcagatcggg gatgccctgt 11
  • the DNA sequence of formula V corresponds to the coding region of human genomic DNA encoding GnT I.
  • Another example of a DNA sequence encoding human GnT I is a larger section
  • the present DNA sequences also include those which may not exactly match the sequences of formulae III-VI, but rather contain a small number of nucleotide substitutions, deletions, and/or additions. Further, the present DNA sequences also include those which encode for amino acid sequences which may not exactly match the sequences of formulae I and II, but rather contain a small number of amino acid residue substitutions, deletions, and/or additions, provided that the protein encoded by the DNA sequence exhibits GnT I activity.
  • the present invention relates to plasmids which contain a DNA sequence encoding rabbit or human GnT I.
  • plasmids may be prepared by conventional techniques and include plasmids formed by inserting one of the present DNA sequences into any suitable plasmid.
  • plasmids include pGEM-7z-rcgntl, in which a 2.5 kb sequence of rabbit cDNA encoding for GnT I ( Figure 2) has been inserted into pGEM-7z; pGEX-2t-rcgntl, in which a 2.5 kb sequence of rabbit cDNA encoding GnT I bas been inserted into pGEX-2t; and pGEM-5z-hggnti, in which a 4 kb sequence of human genomic DNA encoding GnT I has been inserted into pGEM-5z.
  • the present invention relates to transformed microorganisms which contain a heterologous
  • SUBSTITUTE SHEET sequence of DNA encoding rabbit or human GnT I examples include: bacteria, such as E. coli, Brevibacteria, and Coryneforms; fungus, such as Trichoderma reesei, Aspergillus niger, and Aspergillus awamori; yeast, such as Saccharomvees eerevisiae. Candida albicans, Candida utilis, Candida parapsilosis, Schizosaccharomvces pombe, Bandeiraea simplicifolia.
  • bacteria such as E. coli, Brevibacteria, and Coryneforms
  • fungus such as Trichoderma reesei, Aspergillus niger, and Aspergillus awamori
  • yeast such as Saccharomvees eerevisiae.
  • Candida albicans Candida utilis, Candida parapsilosis, Schizosaccharomvces pombe, Bandeiraea simpli
  • the transformed cells may be prepared by transfecting the cells with any of the present plasmids by conventional methods.
  • the present method comprises cell— ree or in vitro expression of one of the present DNA sequences to obtain GnT I.
  • the present method comprises cell— ree or in vitro expression of one of the present DNA sequences to obtain GnT I.
  • in vitro transcription and translation of one of the present plasmids using a system such as described in Methods in Molecular Biology, Nucleic Acids, Walker, ed., Humana Press, Clifton, NJ, pp 145-155 (1984) yields GnT I.
  • the present method comprises' culturing a microorganism which contains a heterologous DNA sequence which corresponds to one of the present DNA sequences.
  • culturing conditions such as time, medium, temperature, light, and agitation, will depend on the identity of the host microorganism and the yield of GnT I desired, these conditions are readily determined by those skilled in the art.
  • the present invention relates to a method for converting a glycoprotein which is in the high mannose form to a glycoprotein which is in the form of a hybrid or complex N-glycan.
  • the present method may be carried out by reacting, in vitro f a glycoprotein which is in the high mannose form with mannosidases followed by UDP-GlcNAc in the presence of GnT I.
  • the present method may comprise culturing a cell which produces a glycoprotein in high mannose form and which also contains a heterologous sequence of DNA encoding human or rabbit GnT I.
  • transfeetion of cell which normally produces a glycoprotein in a mannose form
  • one of the present plasmids may be used to form a cell which produces the protein (produced in high mannose form before transfeetion) as a hybrid or complex N-glycan.
  • the glycoprotein, which is produced in the high mannose form prior to transfeetion with the present DNA is also produced by the host cell as a result of transformation.
  • the DNA encoding the glycoprotein is also heterologous with respect to the host cell.
  • glycoproteins examples include Tanner et al, Biochimica et Biophysica Acta; vol. 906, pp. 81-99 (1987) ; and Kukurazinska et al, Ann. Rev. Biochem. , vol.
  • branched glycans on membrane glycoproteins have been implicated in a variety of biological phenomena, e.g. tumor progression and metastasis, embryogenesis, cell differentiation, cell-cell and receptor-ligand interactions, viral and bacterial infectivity, fertilization and the control of the immune system (Rademacher et al (1988) Ann. Rev. Biochem.. vol. 57, 785-838; Pierce et al (1986) J. Biol. Chem.. vol. 261, 10772-10777; Yamashita et al (1985) J. Biol. Chem., vol. 260, 3963-3969; Schachter (1986) Biochem. Cell Biol.. vol. 64, 163-181; West (1986) Mol. Cell. Biochem.. vol. 72,
  • the remaining transferases share no significant sequence similarities but have very similar domain structures, i.e., a short amino-terminal cytoplasmic tail, a 16-20 amino acid transmembrane segment (non-cleavable signal-anchor domain) , a "stem” or “neck” region of undetermined length, and a long carboxyterminal catalytic domain which is in the Golgi lumen (Paulson et al (1989) J. Biol. Chem.. vol. 264, 17615-17618).
  • the presence of a "neck” region is based on the finding that the ⁇ 2,6-sialyltransferase (Weinstein et al (1987) J. Biol. Chem. vol. 262, 17735-17743; Lammers et al (1988) Biochem. J. , vol. 256, 623-631) and the /31,4-Gal-transferase (D'Agostaro et al (1989) Eur. J. Biochem.. vol. 183, 211-217) can be cut by proteases to release a smaller catalytically active protein lacking the trans-membrane domain.
  • the exact length of this "neck” region cannot be stated with accuracy since it is not known how much of the amino-terminal sequence can be removed without loss of
  • Rabbit GnT I, human, mouse and bovine UDP-Gal:GlcNAc-R 31,4-Gal-transferases and human UDP-GalNAc:Fuc ⁇ l,2Gal-R (GalNAc to Gal) ⁇ l,3-GalNAc-transferase have an abnormally high number of Pro residues between the transmembrane domain and the catalytic domain, e.g., there are 13 Pro residues in GnT I between the transmembrane domain and base position 376 ( Figure 4) ; 9 of these Pro residues occur in a short stretch of 21 amino acids (bases 314-376, Figure 4) .
  • This Pro-rich "neck” may play a role in positioning the catalytic domain in the lumen of the Golgi to enable glycosylation of glycoproteins moving along the Golgi lumen.
  • GnT I The domain structure of GnT I appears to be similar to that of the previously cloned glycosyltransferases. However, GnT I differs from these transferases in being a edial-Golgi enzyme, at least in some tissues (Dunphy et al
  • GnT I Comparison with GnT I reveals a 16-amino acid sequence in GnT I (LHYRPSAELFPIIVSQ, bases 431-478, Figure 4) which shows a high similarity score to amino acid residues 403-418 in ⁇ -mannosidase II (LQYRNYEQLFSYMNSQ) .
  • Paulson's group Paulson et al (1989) J. Biol. Chem., vol. 264, 17615-17618; Colley et al (1989) J. Biol. Chem. , vol.
  • trans-Golgi retention signal lies in the amino-terminal 57 amino acids of the ⁇ 2,6-sialyltransferase molecule.
  • the 16-amino acid "consensus" sequence present in GnT I and ⁇ -mannosidase II may be the equivalent medial-Golgi retention signal. Joziasse et al (1989) J. Biol. Chem. , vol.
  • GnT I was retained on the reversed-phase column under these conditions whereas glycerol, Triton X-100 and salts were washed through the column with 100% n-propanol.
  • GnT I was eluted at 0.1 ml/min as a sharp peak by a linear gradient (5%/min) of decreasing n-propanol concentration (100% to 50%) generated with 100% n-propanol and 50% n-propanol/50% water containing 0.4% (v/v) trifluoroacetic acid at 40°C.
  • GnT I-containing fractions from the inverse gradient RP-HPLC were pooled, adjusted to 0.02% (w/v) with respect to Tween 20 (Pierce Chemical Co., Rockford, IL, USA), concentrated to 100 ⁇ l in a l.5-ml polypropylene tube using a centrifugal vacuum concentrator to reduce the n-propanol concentration, and diluted to 1.5 ml with 5% (v/v) formic acid containing 0.02% Tween 20.
  • GnT I was digested with pepsin (Sigma) at an enzyme/substrate mass ratio of 1:20 for 1 h at 37°C and the digest was fractionated by RP-HPLC on a short microbore column (30 x 2.1 mm i.d.) employing a low pH (trifluoroacetic acid, pH 2.1) mobile phase and a gradient of acetonitrile to yield peptides 5 and 6 ( Figure 1) .
  • RP-HPLC HPLC.
  • RP-HPLC was carried out on a Hewlett-Packard liquid ehromatograph (model 1090A) fitted with a diode array detector (model 1040A) (Simpson et al (1988) Eur, J. Biochem. r vol. 176, 187-197) .
  • a Brownlee RP-300 column (30-nm pore size, 7- ⁇ m diameter dimethyloctylsilica particles packed into a stainless steel cartridge, 30 x 2.1 mm i.d.; Brownlee Laboratories, Santa Clara, CA, USA) was used for all peptide separations.
  • Oligonucleotides and cDNA Synthesis Oligonucleotides and cDNA Synthesis. Oligonucleotides were synthesized on a Pharmacia automated oligonucleotide synthesizer at the Hospital for Sick Children-Pharmacia Biotechnology Service Centre. Total RNA was prepared from rabbit liver by the method of Chirgwin et al (Chirgwin et al
  • TITUTE SHEET Poly(A)+RNA was prepared by oligo(dt) chromatography (Aviv et al (1972) Proc. Natl. Acad. Sci, USA, vol. 69, 1408-1412) using the mRNA Purification Kit supplied by Pharmacia. Single-stranded cDNA synthesis was performed using the RiboClone cDNA Synthesis System (Promega) with the following modifications. Total rabbit liver RNA (20 ⁇ g) in a volume of 5.5 ⁇ l was heated at 65"C for 3 min followed by cooling on ice for 5 min.
  • the following reagents were added to a final volume of 50 ⁇ l:50 mM Tris-HCl, pH 8.3; 0.15 M KC1; 10 mM MgCl 2 ; 2 mM dithiothreitol (DTT) ; each dNTP at 0.4 mM; 40 units of RNasin (Promega) ; 2 mM sodium pyrophosphate; a mixture of the three anti-sense oligonucleotide primers 2A, 3A and 6A ( Figure 1) at concentrations of 50 nM each; 20 units of AMV reverse transcriptase and 15 units of murine leukemia virus reverse transcriptase. Incubation was at 42°C for 2 hr.
  • the reaction mixture was treated with NaOH (0.25 N final concentration) for 5 min at room temperature to destroy RNA.
  • the solution was then heated at 65°C for 1 min followed by cooling on ice for 5 min and neutralized with HC1 (0.25 N final concentration).
  • This cDNA preparation was used directly in the PCR reaction.
  • PCR was carried out in a total volume of 0.1 ml containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.01% gelatin, each of the four dNTP at 0.2 mM, 0.5 ⁇ M of each oligonucleotide in six paired combinations of oligonucleotide primers (2S-3A, 2S-6A, 3S-2A, 3S-6A, 6S-2A, 6S-3A, Figure 1) , 10 ⁇ l of RNA-free rabbit liver cDNA (see above), 2.5 units of Thermus aquaticus (Taq) polymerase (Perkin-Elmer/Cetus) and 0.1 ml of mineral oil.
  • Taq Thermus aquaticus
  • DNA Thermal Cycler Perkin-Elmer
  • DNA Thermal Cycler Perkin-Elmer
  • SUBSTITUTE SHEET Two PCR products (0.45 and 0.50 kb) were detected and were purified from a 1% agarose gel by GeneClean. The DNA ends were filled in with T4 DNA polymerase (Moremen (1989) Proc. Natl. Acad. Sci. USA, vol. 86(14), 5276-5280) and the blunt ends were ligated into Sm ⁇ l site of pGEM-7z (Promega) . The recombinant plasmid was amplified in E. coli XLl-blue cells and purified. The plasmid was used for sequencing and to prepare a labelled probe for screening of a cDNA library.
  • the reaction contained in a total volume of 25 ⁇ l:32 mM Tris-HCl, pH 7.5; 5 mM MgCl 2 ; 2 mM spermidine; 8 mM sodium chloride; 8 mM DTT; 40 units RNasin; 0.4 mM of each of ATP, GTP and UTP; 5 ⁇ l[ ⁇ - 32 P]CTP (800 Ci/mmole) ;
  • RNA probe was desalted over a Sephadex G-50 column (Nick Column, Pharmacia) .
  • a rabbit liver cDNA library in ⁇ gt 10 (5'-stretch. Cat. No TL 1006a from Clontech, EcoRI cloning site) was propagated in E. coli LE392 host cells and IO 6 plaques were screened by standard plaque hybridization techniques (Mania is et al (1982) Molecular Cloning: a laboratory manual. Cold Spring Harbor, N.Y. :Cold Spring Harbor Laboratory) using the above riboprobe. Following fixation of DNA to nitrocellulose membranes, the membranes were washed for 1 hr at 45°C in 50 mM Tris-HCl, pH 8.0/1 M NaCl/1 mM EDTA/0.1% SDS.
  • Membranes were prehybridized at 50°C for 2 hr in 1M NaCl/50 mM sodium phosphate, pH 6.5/0.1% SDS/50% freshly-deionized formamide/1% glycine/0.5% Blotto/5 mM EDTA/1% yeast total RNA. Riboprobe (5 x IO 6 cpm/ml hybridization solution) was added and hybridization was carried out at 50°C overnight. Membranes were washed in 2XSSC/0.1% SDS twice for 5 min at room temperature and twice for 15 min at 50°C. Positive isolates were identified by autoradiography and were plaque-purified.
  • DNA was purified from phage lysates, digested with EcoRI, and cDNA inserts were analyzed by agarose gel electrophoresis. The largest cDNA insert obtained was 1.6 kb; it was subcloned into the EcoRI site of pGEM-7z (Promega) by standard methods (Maniatis et al (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor, N.Y. :Cold Spring Harbor Laboratory) and the recombinant plasmids were transfected into E. coli XLl-blue.
  • Colonies containing the recombinant plasmid were selected and amplified, and plasmid DNA was purified by CsCl gradient centrifugation (Ausubel et al (1990) Current Protocols in Molecular Biology, Media, PA:Greene Publishing Associates and John Wiley and Sons) .
  • the cDNA library was re-screened as described above using a 80 bp riboprobe prepared from the 5'-end of the 1.6 kb clone.
  • the largest cDNA insert obtained was 3.0 kb.
  • This insert was sub-cloned into pGEM-7z as described above and plasmid DNA was purified by CsCl gradient centrifugation (Ausubel et al (1990) Current Protocols in Molecular Biology. Media, PA:Greene Publishing Associates and John Wiley and Sons) , to obtain pGEM-7z-rcgntl.
  • the 1.6 and 3.0 kb clones were sequenced by the Erase-a-Base System (Promega) and the single-strand dideoxynucleotide-chain-termination method. Both DNA strands were sequenced by using colonies in which
  • exonuclease III Erase-a-Base System, Promega
  • Miniplasmid preparations were carried out on about 5-10 subclones from each exonuclease III time point and were cut with BamHl and Aatll to determine DNA size. Colonies with appropriate deletions were amplified and incubated with M13K07 helper phage at 37°C for 1 hr followed by amplification in the presence of kanamycin (70 ⁇ g/ml) for 6 hr at 37°C. Single-stranded DNA was produced by the helper phage and excreted into the medium.
  • the ss-DNA was purified from the medium by polyethylene glycol precipitation and sequenced by the dideoxynucleotide chain-termination method using deoxyadenosine 5'-[ ⁇ -[ 35 S]thio]triphosphate, Sequenase (United States Biochemical) and the forward primer for pGEM-7z.
  • RNA Hybridization Rabbit liver poly(A)+RNA (5 ⁇ g) was denatured in 50% (v/v) formamide/6% (v/v) formaldehyde buffer at 65°C and was resolved by gel electrophoresis in a 1% agarose gel containing 6% (v/v) formaldehyde. The RNA was transferred to a nitrocellulose filter and the filters were hybridized with the 32 P-labelled 0.5 kb PCR riboprobe (see above) followed by autoradiography. The specific activity of the probe was about 10 s dpm/ng and the hybridization solution contained about 10 6 dpm/ml.
  • RNA synthesis was carried out at 40°C for 1 hr in a total volume of 50 ⁇ l containing 40 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 , 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 40 units RNasin (Promega), 0.5 mM of each of ATP, UTP and CTP, 0.1 mM GTP, 0.5 mM m 7 G(5')PPP(5 » )G (Pharmacia), 10 units SP6 RNA polymerase and 10 ⁇ g linearized plasmid.
  • Control incubations were carried out in the absence of plasmid or with a linearized pGEM-7z recombinant plasmid containing a non-coding insert.
  • the reaction mixture was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1, v/v) followed by precipitation with cold ethanol.
  • Protein synthesis was carried out at 30°C for 1 hr in a total volume of 50 ⁇ l containing all 20 amino acids (1 mM each) , 20 units of RNasin, RNA as prepared above, and buffer and rabbit reticulocyte lysate as supplied by Promega (Olliver et al (1984) "In vitro translation of messenger RNA in a rabbit reticulocyte lysate cell-free system", in: M. Walker J., ed. , Methods in Molecular Biology, Nucleic Acids, Clifton, N.J. :Humana Press, 145-155) .
  • Non-radioactive amino acids were used when the products of translation were assayed for GnT I activity (see below) . Separate incubations were carried out with L-[ 35 S]-methionine (1000 Ci/mmole; 90 ⁇ Ci/incubation) replacing non-radioactive Met; these incubations were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
  • GnT I was assayed (Schachter (1989) Methods Enzymol. , vol. 179, 351-396; Brockhausen et al (1988) Biochem. Cell Biol. , vol. 66, 1134-1151) in a total volume of 40 ⁇ l containing 20 mM MnCl 2 , bovine serum albumin (1 mg/ml) , 0.1% (v/v) Triton X-100, 0.1 M MES (pH 6.1), 0.5 mM UDP-N-[1- 14 C]acetyl-D-glucosamine (2.2 mCi/mmole) , 0.125 M GlcNAc and 0.6 mM Man ⁇ l-6(Man ⁇ l-3)Man?-hexyl (a kind gift from Dr.
  • SUBSTITUTE SHEET (Cl-form, 100-200 mesh, equilibrated with water) to remove radioactive nucleotide-sugar.
  • the eluate was applied to a Sep-Pak C-18 reverse phase cartridge (Waters) conditioned with 20 ml methanol and 20 ml water.
  • the cartridge was washed with 20 ml water and radioactive product was eluted with 5.0 ml methanol (Palcic et al (1988) Glycocon uguate J. , vol. 5, 49-63) .
  • pGEX-2t-rcgntl This plasmid was prepared from pGEM-7z-rcgntl by cutting out the insert rcgntl with Eco RI. Plasmid pGEX-2t (Pharmacia) was linearized with Eco RI and the insert was ligated into the plasmid by standard procedures. The recombinant plasmid was amplified in E. coli in the presence of ampicillin and purified by cesium chloride centrifugation.
  • PCR was carried out with all six possible combinations of sense and anti-sense primers (2S-3A, 2S-6A, 3S-2A, 3S-6A, 6S-2A, 6S-3A, Figure 1) .
  • the products of the PCR reactions were analyzed by agarose gel electrophoresis ( Figure 3) .
  • Primer-dependent products were obtained with two of the six incubations, i.e., 2S-6A (500 bp) and 3S-6A (450 bp) .
  • the complete nucleotide sequence for GnT I is shown in Figure 4.
  • HEET Oligonucleotide primers 2S and 3A are separated by only nine bases thereby explaining the absence of PCR product with this combination.
  • the 1.6 kb clone contains 0.5 kb from the 3'-end of the coding region and the full 1.1 kb 3 '-untranslated region (rcl600, Figure 2).
  • the 3.0 kb clone yielded a 2485 bp sequence (rc2500, Figure 2; Figure 4).
  • rc2500 Figure 2; Figure 4
  • subcloning of the 3.0 kb DNA fragment in pGEM-7z results in deletion of a 0.5 kb DNA fragment near the 5'-end of the clone.
  • Comparison of the cDNA sequence shown in Figure 4 with the sequence of human genomic DNA for GnT I (in preparation) has shown that this deleted 0.5 kb DNA fragment is not part of the GnT I gene; we do not know the origin of this DNA.
  • the GnT I coding sequence has 1341 bp and codes for a membrane-bound protein of 447 amino acids (M r 52,000). There is a single hydrophobie domain (bases 62 to 136) flanked by charged amino acids ( Figure 4) . Chou-Fasman rules (Chou et al (1978) Adv. Enzvmol.. vol. 47, 45-147) predict that this hydrophobie segment is capable of propagating an ⁇ -helix, as expected for a transmembrane domain.
  • the presumptive initiation Met codon is at the ATG ' codon at position 50 which has an A at position 47 thereby fulfilling the requirements for an initiation codon (Kozak (1983) Microbiological Reviews, vol. 47, 1-45). All eight peptides shown in Figure 1 (a total of 103 amino acid residues) can be identified in the sequence ( Figure 4) ; an additional five tentative assignments also match the sequence. GnT I purified from rabbit liver has a molecular weight of about 45 kDa (Nishikawa et al (1988) J. Biol. Chem. r vol. 263, 8270-8281).
  • the protein has no N-glycans since none of the nine Asn residues are in a typical Asn-X-Ser(Thr) sequence; we have previously shown that rabbit liver GnT I binds poorly to lectin/agarose columns (Nishikawa et al (1988) J. Biol. Chem. , vol. 263, 8270-8281) . If there are no or few 0-glycans, a
  • SUBSTITUTE SHEET catalytically active protein of 45 kDa can be derived by cleavage at about base position 215 ( Figure 4) .
  • the complete sequence has a long 3'-untranslated region (bases 1391-2479) containing the consensus polyadenylation signal AATAAA at position 2435 (Tosi et al (1981) Nucleic Acids Research, vol. 9, 2313-2323). Long 3'-untranslated regions are typical of the known glycosyltransferase genes and may be a feature present in other Golgi-localized enzymes (Moremen (1989) Proc. Natl. Acad. Sci. USA, vol. 86(14), 5276-5280).
  • Northern Blot Analysis The PCR riboprobe was used to determine the size of mRNA in rabbit liver. A major band was detected at about 3.0 kb with some smearing at lower molecular weights (data not shown) indicating that the 2.5 kb cDNA clone ( Figure 4) may not be full-length.
  • PCR polymerase chain reaction
  • the rabbit cDNA probe was used to screen 10 s plaques from an amplified human genomic DNA library in ⁇ EMBL3 prepared from chromosomal DNA from chronic yeloid leukemia cells. Positive plaques (23) were purified and phage DNA was subjected to restriction enzyme analysis using the 0.5 kb rabbit cDNA as probe. All 23 preparations gave the same Sau3A 0.4 kb fragment. This fragment showed 87% base similarity and 90% amino acid sequence similarity to the rabbit GnT I carboxy-terminal sequence. Inserts of 13 and 15 kb were cut from two of the human genomic DNA clones with SAII and subcloned into plasmid pGEM-5zf(+) (Promega) . Restriction maps of the two inserts show that they represent an over-lapping 18 kb DNA sequence.
  • the coding sequence was located in a 4.0 kb fragment of human genomic DNA by screening restriction maps with a probe containing the entire coding region of the rabbit GnT I cDNA. This 4.0 kb DNA fragment was cut out by restriction enzymes and subcloned into the sequencing vector pGEM-5zf(+) to yield pGEM-5z-hggntl and sequenced. Transfeetion of the gene into Lee 1 Chinese hamster ovary cell mutants (which lack GnT I activity) results in the expression of GnT I activity indicating the presence of a functional promoter 5'-upstream of the transcription start site.
  • the 4 kb sequence contains an open reading frame coding for a protein with 445 amino acids (2 less than the rabbit enzyme) .
  • the DNA contains a functional promoter and an intronless gene.
  • the similarity between the rabbit and human enzymes is 85% for the nucleotide coding sequences and over 90% for the amino acid sequences.

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Abstract

On a cloné les gènes codant la GnT I de l'homme et du lapin.
PCT/CA1991/000417 1990-11-30 1991-11-29 CLONAGE DE UDP-N-ACETYLGLUCOSAMINE:α-3-D-MANNOSIDE β-1,2-N-ACETYLGLUCOSAMINYLTRANSFERASE I WO1992009694A2 (fr)

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* Cited by examiner, † Cited by third party
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EP0585109A2 (fr) * 1992-08-24 1994-03-02 Suntory Limited N-acétylglucosaminyl transférase, gène codant pour cela, vecteurs et hôtes transformés correspondants et procédé de production de cette transférase
EP0585083A1 (fr) * 1992-08-21 1994-03-02 Takara Shuzo Co. Ltd. Gène de glycosyltransferase humain
US5874271A (en) * 1992-08-21 1999-02-23 Takara Shuzo Co., Ltd. Human glycosyltransferase gene, compounds and method for inhibiting cancerous metastasis
WO1999029879A1 (fr) * 1997-12-09 1999-06-17 Antje Von Schaewen SEQUENCES DE GntI VEGETALES ET UTILISATION DE CELLES-CI POUR L'OBTENTION DE PLANTES NE PRESENTANT PAS UNE ACTIVITE DE N-ACETYLGLUCOSYLAMINYLTRANSFERASE I GnTI OU BIEN SEULEMENT UNE TELLE ACTIVITE REDUITE
WO2004003194A3 (fr) * 2002-06-26 2004-04-22 Flanders Interuniversity Inst Modification de la glycosylation des proteines chez pichia pastoris
EP1715057A2 (fr) * 1994-12-30 2006-10-25 AB Enzymes GmbH Méthodes de modification d'hydrates de carbone
US7507573B2 (en) 2003-11-14 2009-03-24 Vib, Vzw Modification of protein glycosylation in methylotrophic yeast
US8986949B2 (en) 2003-02-20 2015-03-24 Glycofi, Inc. Endomannosidases in the modification of glycoproteins in eukaryotes
US9187552B2 (en) 2010-05-27 2015-11-17 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US9328170B2 (en) 2011-05-25 2016-05-03 Merck Sharp & Dohme Corp. Method for preparing Fc containing polypeptides having improved properties

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* Cited by examiner, † Cited by third party
Title
Biochem. Soc. Trans., vol. 19, no. 3, August 1991, Biochemical Society, (London, GB), H. SCHACHTER et al.: "Molecular cloning of human and rabbit UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I", pages 645-648, see page 646, left-hand column, line 1 - page 648, right-hand column, line 23 *
Glycoconjugate Journal, vol. 7, no. 5, 10 October 1990, (Lund, SE), E. HULL et al.: "Isolation of 13 and 15 kilobase human genomic DNA clones containing the gene for UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I", page 468, abstract no. 85, see the whole document *
Glycoconjugate Journal, vol. 7, no. 5, 10 October 1990, (Lund, SE), M. SARKAR et al.: "Rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I: characteriazation of a 2,5 kilobase cDNA clone", page 380, abstract no. 4, see the whole document *
J. Biol. Chem., vol. 256, no. 2, 25 January 1981, Am. Soc. Biol. Chem., Inc., (US), C.L. OPPENHEIMER et al.: "Purification and characterization of a rabbit liver alpha1 3 mannoside beta1 2 N-acetylglucosaminyltransferase", pages 799-804, see page 801, left-hand column, line 8 - right-hand column, line 8 (cited in the application) *
J. Biol. Chem., vol. 263, no. 17, 15 June 1988, Am. Soc. Biol. Chem., Inc., (US), Y. NISHIKAWA et al.: "Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I", pages 8270-8281, see table I; abstract; page 8270, right-hand column, lines 25-29 (cited in the application) *
J. Biol. Chem., vol. 265, no. 2, 15 January 1990, Am. Soc. Biol. Chem., Inc., (US), F. YAMAMOTO et al.: "Cloning and characterization of DNA complementary to human UDP-Ga1NAc: Fucalpha1 2Ga1 alpha1 3Ga1NAc transferase (histo-blood group A transferase) mRNA", pages 1146-1151, see materials and methods (cited in the application) *
Proc. Natl. Acad. Sci. USA, vol. 88, no. 1, January 1991, Natl. Acad. Sci., (Washington, DC, US), M. SAKKAR et al.: "Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I", pages 234-238, see figure 4; page 236, left-hand column, line 26 - page 237, right-hand column, line 6 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0585083A1 (fr) * 1992-08-21 1994-03-02 Takara Shuzo Co. Ltd. Gène de glycosyltransferase humain
US5874271A (en) * 1992-08-21 1999-02-23 Takara Shuzo Co., Ltd. Human glycosyltransferase gene, compounds and method for inhibiting cancerous metastasis
US5876714A (en) * 1992-08-21 1999-03-02 Takara Shuzo Co., Ltd. Human glycosyltransferase gene, compounds and method for inhibiting cancerous metastasis
EP0585109A2 (fr) * 1992-08-24 1994-03-02 Suntory Limited N-acétylglucosaminyl transférase, gène codant pour cela, vecteurs et hôtes transformés correspondants et procédé de production de cette transférase
EP0585109A3 (en) * 1992-08-24 1994-07-06 Suntory Ltd N-acetylglucosaminyl transferase, gene coding therefor, corresponding vectors and transformed hosts, processes for production thereof
US5707846A (en) * 1992-08-24 1998-01-13 Suntory Limited N-acetylglucosaminyl transferase gene coding therefor and process for production thereof
US5834284A (en) * 1992-08-24 1998-11-10 Suntory Limited N-acetylglucosaminyl transferase gene coding therefor and process for production thereof
EP1715057A2 (fr) * 1994-12-30 2006-10-25 AB Enzymes GmbH Méthodes de modification d'hydrates de carbone
WO1999029879A1 (fr) * 1997-12-09 1999-06-17 Antje Von Schaewen SEQUENCES DE GntI VEGETALES ET UTILISATION DE CELLES-CI POUR L'OBTENTION DE PLANTES NE PRESENTANT PAS UNE ACTIVITE DE N-ACETYLGLUCOSYLAMINYLTRANSFERASE I GnTI OU BIEN SEULEMENT UNE TELLE ACTIVITE REDUITE
US6653459B1 (en) 1997-12-09 2003-11-25 Antje Von Schaewen Plant GntI sequences and the use thereof for the production of plants having reduced or lacking N-acetyl glucosaminyl transferase I(GnTI) activity
US8883445B2 (en) 2002-06-26 2014-11-11 Research Corporation Technologies, Inc. Protein glycosylation modification in methylotrophic yeast
WO2004003194A3 (fr) * 2002-06-26 2004-04-22 Flanders Interuniversity Inst Modification de la glycosylation des proteines chez pichia pastoris
US7252933B2 (en) 2002-06-26 2007-08-07 Flanders Interuniversity Institute For Biotechnology Protein glycosylation modification in methylotrophic yeast
AU2003238051B2 (en) * 2002-06-26 2008-03-13 Research Corporation Technologies, Inc. Protein glycosylation modification in pichia pastoris
US8986949B2 (en) 2003-02-20 2015-03-24 Glycofi, Inc. Endomannosidases in the modification of glycoproteins in eukaryotes
US8058053B2 (en) 2003-11-14 2011-11-15 Vib, Vzw Modification of protein glycosylation in methylotrophic yeast
US7507573B2 (en) 2003-11-14 2009-03-24 Vib, Vzw Modification of protein glycosylation in methylotrophic yeast
US9187552B2 (en) 2010-05-27 2015-11-17 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US10858686B2 (en) 2010-05-27 2020-12-08 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US11959118B2 (en) 2010-05-27 2024-04-16 Merck Sharp & Dohme Llc Fc-containing polypeptides having improved properties and comprising mutations at positions 243 and 264 of the Fc-region
US9328170B2 (en) 2011-05-25 2016-05-03 Merck Sharp & Dohme Corp. Method for preparing Fc containing polypeptides having improved properties

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