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WO1999011796A1 - Sulfotransferase hnk-1 et ses methodes d'utilisation - Google Patents

Sulfotransferase hnk-1 et ses methodes d'utilisation Download PDF

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Publication number
WO1999011796A1
WO1999011796A1 PCT/US1998/018572 US9818572W WO9911796A1 WO 1999011796 A1 WO1999011796 A1 WO 1999011796A1 US 9818572 W US9818572 W US 9818572W WO 9911796 A1 WO9911796 A1 WO 9911796A1
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Prior art keywords
sulfotransferase
hnk
cell adhesion
neural
sequence
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PCT/US1998/018572
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English (en)
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Ned Mantei
Hendrikus Bakker
Melitta Schachner
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Acorda Therapeutics
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Application filed by Acorda Therapeutics filed Critical Acorda Therapeutics
Priority to EP98946870A priority Critical patent/EP1012300A1/fr
Priority to AU93791/98A priority patent/AU9379198A/en
Publication of WO1999011796A1 publication Critical patent/WO1999011796A1/fr

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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.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates generally to the modulation of neural growth and immune system derived cells in immune responses under normal and pathological conditions, in the nervous system, and more particularly to methods and associated agents, constructs and compositions for improving neural growth.
  • the invention relates to the cloning and expression of the HNK-1 sulfotransferase gene, and production of the recombinant protein for use with neural cell adhesion molecules such as LI , to foster and improve such neural growth.
  • Glial cells are the decisive determinants for controlling axon regrowth. Mammalian glial cells are generally permissive for neurite outgrowth in the central nervous system during development (Silver et al. (1982) J. Comp. Neurol. 210: 10-29; Miller et al. (1985) Develop. Biol. 111:35-41; Pollerberg et al. (1985) 7. Cell. Biol. 101: 1921-1929) and in the adult peripheral nervous system (Fawcett et al. (1990) Ann . Rev. Neurosci. 13:43-60).
  • glial cells of the adult mammalian peripheral nervous system can revert to some extent to their earlier neurite outgrowth-promoting potential, allowing them to foster regeneration (Kalderon (1988) J. Neurosci Res. 21:501-512; Kliot et al. "Induced regeneration of dorsal root fibers into the adult mammalian spinal cord, " In: Current Issues in Neural Regeneration, New York, pp. 311-328; Carlstedt et al. (1989) Brain Res. Bull. 22:93-102). Glial cells of the central nervous system of some lower vertebrates remain permissive for neurite regrowth in adulthood (Stuermer et al. (1992) J. Neurobiol. 23:537-550). In contrast, glial cells of the central nervous system of adult mammals are not conducive to neurite regrowth following lesions.
  • LI Within two days LI accumulates at sites of contact between neurons and Schwann cells being concentrated mainly at the cell surface of Schwann cells but not neurons (Martini et al. (1994a)). Furthermore, the homophilic binding ability of LI is enhanced by molecular association with the neural cell adhesion molecule N-CAM, allowing binding to occur through homophilic assistance (Kadmon et al. (1990a); Kadmon et al. (1990b) J. Cell Biol. 110:209-218 and 110: 193-208; Horstkorte et al. (1993) J. Cell. Biol. 121 : 1409- 1421).
  • LI Besides its neurite outgrowth promoting properties, LI also participates in cell adhesion (Rathjen et al. (1984) EMBO J. 3: 1-10; Kadmon et al. (1990b) J. Cell. Biol. 110:209-218; Appel et al. (1993) 7. Neurosci. , 13:4764-4775), granule cell migration (Lindner et al. (1983) Nature 305:427-430) and myelination of axons (Wood et al. (1990) J. Neurosci 10:3635-3645).
  • LI consists of six immunoglobulin-like domains and five fibronectin type III homologous repeats. LI acts as a signal transducer, with the recognition process being a first step in a complex series of events leading to changes in steady state levels of intracellular messengers. The latter include inositol phosphates, Ca 2 *, pH and cyclic nucleotides (Schuch et al. (1990) Neuron 3:13-20; von Bohlen und Hallbach et al. (1992) Eur. J. Neurosci. 4:896-909; Doherty et al. (1992) Curr. Opin. Neurobiol.
  • Ll-mediated neurite outgrowth is sensitive to the blockage of L type Ca 2+ channels and to pertussis toxin.
  • LI is also present on proliferating, immature astrocytes in culture and neurite outgrowth is promoted on these cells far better than on differentiated, LI immunonegative astrocytes (Saad et al. (1991) 7. Cell. Biol. 115:473-484). In vivo, however, astrocytes have been found to express LI at any of the developmental stages examined from embryonic day 13 until adulthood (Bartsch et al. (1989) 7. Comp. Neurol 284:451-462; and unpublished data).
  • the carbohydrate antigen recognized by the monoclonal antibody HNK-1 was originally described as a marker for human natural killer cells (Abo and Balch, (1981) 7. Immunology 127:1024-1029). Later it was shown to be expressed predominantly on glycolipids and glycoproteins from nervous tissue (McGarry et al, (1983) Nature 306:376-378; Ilyas et al, (1984) Biochem. Biophys. Res. Comm. 122:1206-1211; Kruse et al. , (1984) Nature 311:153-155; Yuen et al., (1997) 7. Biol. Chem. 272:8924-8931).
  • HNK-1 carbohydrate in both the central and peripheral nervous system is spatially and developmentally regulated (Wernecke et al., (1985) 7. Neuroimmunol. 9:115-130; Holley and Yu (1987) Dev. Neurosci. 9: 105-19; Prasadarao et al., (1990) 7. Neurochem. 55:2024- 2030; Chou et al., (1991) J. Neurochem. 57:852-859; Low et al. , (1994) Eur. J. Neurosci. 6:1773-1781; Jungalwala (1994) Neurochem. Res. 19:945-957).
  • the HNK-1 carbohydrate epitope is carried by many, but not all, neural recognition glycoproteins, and is involved in homo- and heterophilic binding of these proteins (for a review, see Schachner and Martini (1995) Trends Neurosci. 18:183-191).
  • Of special interest is the association of the epitope with Schwann cells myelinating motor but not sensory axons (Low et al., (1994) Eur. J. Neurosci. 6:1773-1781), where it may be involved in the preferential reinnervation of muscle nerves by motor axons after lesion (Martini et al., (1992) Eur. J. Neurosci. 4:628-639; Martini et al., (1994) 7. Neurosci. 14:7180-7191).
  • HNK-1 carbohydrates The key enzymes in the biosynthesis of HNK-1 carbohydrates are a glucuronyltransferase (Chou et al, (1991) J. Biol Chem. 266: 17941-17947; Oka et al, (1992) 7. Biol. Chem. 267:22711-22714), transferring GlcA in ⁇ l-3 linkage to a terminal galactose, and a sulfotransferase (Oka et al, (1992) 7. Biol. Chem. 267:22711-22714) responsible for coupling sulfate to the C-3 position of this GlcA residue.
  • a cDNA encoding the glucuronyltransferase involved in the biosynthesis of at least the HNK-1 glycoprotein epitope has recently been cloned (Terayama et al, (1997) Proc. Natl. Acad. Sci. U.S.A. 94:6093-6098; SEQ ID NO:3).
  • the instant invention describes the cloning of a cDNA coding for a sulfotransferase active on terminal glucuronic acid residues, and whose expression can render cells immunoreactive with HNK-1 antibody when cotransfected with a glucuronyltransferase cDNA.
  • Use of this cDNA to generate the HNK-1 glycosylated recombinant Ll-Fc fusion protein can thus facilitate various therapeutic methods where LI, or other members of its immunoglobulin superfamily, can be utilized, especially in the treatment of damaged or diseased neurons. It is toward such objectives that the present invention is directed.
  • the present invention concerns the isolation of a novel sulfotransferase cDNA via an expression cloning strategy.
  • the clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein.
  • the enzyme When expressed as a soluble fusion protein, the enzyme is able to transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.
  • the isolated sulfotransferase can be utilized to prepare proteins having the HNK-1 carbohydrate epitope which is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions.
  • the structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc.
  • the glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope, because the rest of the structure occurs often in glycoco ⁇ jugates.
  • the instant invention thus also provides a method for sulfonation of glucuronic acid, present on cell adhesion and neural cell adhesion molecules, which comprises exposure of the neural cell adhesion molecule to a recombinant sulfotransferase enzyme to generate the HNK-1 carbohydrate epitope thereon.
  • a method for sulfonation of glucuronic acid, present on cell adhesion and neural cell adhesion molecules which comprises exposure of the neural cell adhesion molecule to a recombinant sulfotransferase enzyme to generate the HNK-1 carbohydrate epitope thereon.
  • Use of isolated or recombinant cDNA to generate the HNK-1 glycosylated recombinant Ll-Fc fusion protein can thus facilitate various therapeutic methods where LI , or other members of its immunoglobulin superfamily, can be utilized, especially in the treatment of damaged or diseased neurons.
  • It is a further object of the present invention to provide a method for promoting neural growth in vivo in the nervous system of a mammal comprising administering to said mammal a neural growth promoting amount of an agent, said agent comprising a neural cell adhesion molecule, which molecule is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth, active fragments thereof, secreting cells thereof and soluble molecules thereof, said agent being modified by recombinant means to contain the HNK-1 carbohydrate epitope thereon.
  • FIGURE 1 shows the immunostaining of transiently transfected CHOP2 cells.
  • Panels A to D and F show staining with antibody HNK-1, panel E with L2-412.
  • Cotransfection with the glucuronyltransferase cDNA (SEQ ID NO: 3) and pools of 5,000-10,000 clones from the primary library gave a few immunopositive cells with one pool ⁇ A).
  • Progressively higher frequencies of positive cells were found upon two rounds of subdividing positive pools ⁇ B and Q, reaching a maximum with the single sulfotransferase cDNA clone (TJ).
  • E Cells transfected only with glucuronyltransferase cDNA.
  • F Cells transfected with the sulfotransferase but not glucuronyltransferase cDNA clone.
  • FIGURE 2 shows Western blot analysis of proteins from transfected CHOP2 cells. Blots were stained with antibody HNK-1 ⁇ A) or L2-412 ⁇ B). Cells were transfected with no DNA ⁇ lanes 1), with the glucuronyltransferase cDNA alone ⁇ lanes 2), with sulfotransferase cDNA alone ⁇ lanes 3), or with both transferase cDNAs ⁇ lanes 4).
  • FIGURE 3 shows in vitro sulfotransferase assays. Homogenates were incubated with [ 35 S]-PAPS and different potential acceptor substrates. The reaction products were analyzed by HPTLC.
  • A Cerebral cortex homogenate.
  • B Mock transfected CHOP2 cells.
  • C CHOP2 cells transfected with the sulfotransferase cDNA clone.
  • Lanes 1 for , B, and Q: no acceptor; lanes 2: Gal ⁇ -pNP; lanes 3: GlcA ⁇ -pNP lanes 4: GlcA ⁇ l ⁇ 3Gal ⁇ -R. The bands closest to the origin comigrate with [ 35 S]-PAPS.
  • IgG beads were incubated with medium from cells expressing either the nonsecreted form of the sulfotransferase, without protein A ⁇ lane 1), the protein A fusion protein ⁇ lane 2), or the translation product from a pPROTA vector containing the sulfotransferase in antisense orientation ⁇ lane 3).
  • FIGURE 4 shows the complete nucleotide and deduced amino acid sequence of the HNK-1 -sulfotransferase cDNA clone.
  • the putative transmembrane region in the translation product is underlined and potential N-linked glycosylation sites are indicated by asterisks.
  • the clone finally isolated predicts a polypeptide enzyme of 356 amino acids, with characteristics of a type II transmembrane protein and characterized by the amino acid residue sequence of SEQ ID NO:2.
  • the enzyme When expressed as a soluble fusion protein, the enzyme is able to transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.
  • the isolated sulfotransferase enzyme can be utilized to prepare proteins having the HNK-1 carbohydrate epitope which is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions.
  • the structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcA ⁇ l ⁇ 3Gal ⁇ l-4GlcNAc.
  • the glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope, because the rest of the structure occurs often in glycoconjugates.
  • the instant invention also provides a method for sulfation of glucuronic acid, present on cell adhesion and neural cell adhesion molecules, which comprises exposure of the neural cell adhesion molecule to a recombinant sulfotransferase enzyme to generate the HNK-1 carbohydrate epitope thereon.
  • a method for sulfation of glucuronic acid, present on cell adhesion and neural cell adhesion molecules which comprises exposure of the neural cell adhesion molecule to a recombinant sulfotransferase enzyme to generate the HNK-1 carbohydrate epitope thereon.
  • Use of isolated or recombinant cDNA to generate the HNK-1 glycosylated recombinant Ll-Fc fusion protein can thus facilitate various therapeutic methods where LI, or other members of its immunoglobulin superfamily, can be utilized, especially in the treatment of damaged or diseased neurons.
  • the present invention extends to a novel sulfotransferase having the following characteristics: it exhibits the characteristics of a type II transmembrane protein; and, when expressed as a soluble fusion protein, the enzyme is able to transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.
  • novel sulfotransferases of the present invention have an amino acid residue sequence as represented by SEQ ID NO:2, or a conservative variant thereof.
  • the present invention relates to all members of the herein disclosed family of novel sulfotransferases.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, a degenerate variant thereof, or a sequence hybridizable thereto, which encodes a novel sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule has a nucleotide sequence or is complementary to a DNA sequence as shown in FIGURE 4 (SEQ ID NO:l).
  • the rat DNA sequence of the sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the sulfotransferase.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes the preparation of plasmids including such vectors.
  • the present invention also includes the novel sulfotransferase proteins having the activities noted herein, and that display the amino acid residue sequences set forth and described above and selected from SEQ ID NO: 2, and conservative variants thereof.
  • the instant invention provides a cloned enzyme having no sequence similarity to known proteins.
  • the cloned 2647 bp cDNA fragment contains an open reading frame encoding a protein of 356 amino acids ( Figure 4).
  • the protein appears to be a type II transmembrane protein as a potential transmembrane region is observed close to the N-terminus, and the enzymatic activity is expected to be located in the E.R. or Golgi lumen.
  • No significant sequence similarity was observed between the translation product of the cloned cDNA and any known protein.
  • More than 10 overlapping human ESTs were found that potentially encode the human paralog of the cloned rat sulfotransferase.
  • Several other ESTs showed a much lower similarity with the rat cDNA clone and may encode two different paralogs of the human gene, indicating that there probably is a human gene family of at least three members.
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present sulfotransferase(s), and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO:l, and degenerate variants thereof.
  • a recombinant expression system is provided to produce biologically active animal or human sulfotransferase capable of generating the HNK- 1 carbohydrate epitope on any neural cell adhesion molecule.
  • the present invention naturally contemplates several means for preparation of the sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the isolation of the cDNA and amino acid sequences disclosed herein facilitates the reproduction of the sulfotransferase by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • the invention includes an assay system for screening of potential drugs effective to modulate the sulfotransferase activity of target mammalian cells by interrupting or potentiating the sulfotransferase.
  • the test drug could be administered to a cellular sample with the ligand that activates the sulfotransferase, or an extract containing the activated sulfotransferase, to determine its effect upon the binding activity of the sulfotransferase to any chemical sample (including DNA), or to the test drug, by comparison with a control.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the sulfotransferase and/or factors or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating the sulfotransferase activity.
  • Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.
  • drugs might be used to further enhance the production of cellular adhesion molecules having the HNK-1 carbohydrate epitope thereon, and thereby to further enhance the neurite growth and/or remyelinating and/or neuroprotecting capabilities of such neural cell adhesion molecules.
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the sulfotransferase(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the enhancement of the activity of cellular adhesion molecules, particularly those such as LI , in the treatment of diseased and damaged neurons of the nervous system, and comprises administering the sulfotransferase so as to generate the HNK-1 carbohydrate epitope on the neural cell adhesion molecule.
  • sulfotransferase capable of transferring sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid
  • sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in FIGURE 4 (SEQ ID NO:2), and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated.
  • modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits.
  • sulfotransferase “sulfotransferase capable of transferring sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid,” and “sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D” isomeric form can be substituted for any L- amino acid residue, as long as the desired functional enzymatic activity is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a “replicon” is any genetic element ⁇ e.g. , plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, ter alia, in linear DNA molecules ⁇ e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5 ' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 ' (amino) terminus and a translation stop codon at the 3' (carboxy 1) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic ⁇ e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75 % (preferably at least about 80% , and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al , supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • DNA sequences encoding sulfotransferase(s) which code for a sulfotransferase having the same amino acid sequence as SEQ ID NO:2, but which are degenerate to SEQ ID NO: l .
  • degenerate to is meant that-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in SEQ ID NO:l such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non- conservative manner ⁇ i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner ⁇ i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Another grouping may be those amino acids with phenyl groups:
  • Another grouping may be according to molecular weight (i.e. , size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e. , His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80% , and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature ⁇ e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab' , F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well- known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal ⁇ e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T m with washes of higher stringency, if desired.
  • the present invention concerns the identification of a novel sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule.
  • the present invention relates to all members of the herein disclosed sulfotransferases capable of generating the HNK-1 carbohydrate epitope on any neural cell adhesion molecules, especially LI.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a sulfotransferase capable of generating the HNK-1 carbohydrate epitope on a neural cell adhesion molecule by transferring sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid, or a fragment thereof, that possesses a molecular weight of about 42 kD (up to 51 kD with glycosylation) and an amino acid sequence set forth in FIGURE 4 (SEQ ID NO: 2); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the sulf
  • the possibilities both diagnostic and therapeutic that are raised by the existence of the sulfotransferase derive from the fact that the enzyme appear to participate in direct and causal protein-protein interaction between the sulfotransferase and neural cell adhesion molecules and is capable of generating the HNK-1 carbohydrate epitope on the neural cell adhesion molecule by transferring sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the sulfotransferase is implicated, to modulate the activity initiated by any neural cell adhesion molecule, especially LI.
  • the sulfotransferase or their binding partners or other ligands or agents exhibiting mimicry to the sulfotransferase or control over their production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient where diseased or damaged neurons of the nervous system are under treatment.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the sulfotransferase(s) or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the sulfotransferase(s) and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring the levels thereof.
  • the sulfotransferase or its subunits may be used to produce both polyclonal and monoclonal antibodies to a variety of by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic the activity (ies) of the sulfotransferase(s) of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett et al , “Monoclonal Antibodies” (1980); see also U.S. Patent Nos.
  • Panels of monoclonal antibodies produced against the sulfotransferase(s) can be screened for various properties; i.e., isotype, epitope, affinity, etc.
  • monoclonal antibodies that neutralize the activity of the sulfotransferase or its subunits can be readily identified in sulfotransferase activity assays.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant sulfotransferase is possible.
  • the anti-sulfotransferase antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti- sulfotransferase antibody molecules used herein be in the form of Fab, Fab' , F(ab'), or F(v) portions of whole antibody molecules.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a sulfotransferase, such as an anti- sulfotransferase antibody, preferably an affmity-purified polyclonal antibody, and more preferably a mAb.
  • a sulfotransferase such as an anti- sulfotransferase antibody, preferably an affmity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-sulfotransferase antibody molecules used herein be in the form of Fab, Fab' , V ⁇ b' or F(v) portions or whole antibody molecules.
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a sulfotransferase-binding portion thereof, or sulfotransferase, or an origin-specific DNA-binding portion thereof.
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present sulfotransferase and their ability to inhibit specified sulfotransferase activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • DMEM Dulbecco's minimal essential medium
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a sulfotransferase, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartar ic, mandelic, and the like. Salts formed from the free carboxy 1 groups can also be derived from inorganic bases such as, for example, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of enhancement of the neural cell adhesion molecule's activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0J to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • the therapeutic compositions may further include an effective amount of the neural cell adhesion molecule and/or a glucuronyltransferase (SEQ ID NO: 4) capable of transferring GlcA in Bl 3 Gal Bl 4GlcNAc.
  • a glucuronyltransferase SEQ ID NO: 4
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul means microliter
  • ml means milliliter
  • 1 means liter.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • the present invention therefore comtemplates and includes the promotion of the expression of the sulfotransferase epitope by transfection of cells as by gene therapy techniques known in the art.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g.
  • yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the cytomegalovirus enhancer /promoter, the promoter of the polypeptide chain of elongation factor l gene, the early or late promoters of the, the SV40 or adenovirus system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase ⁇ e.g., Pho5), the promoters of the yeast ⁇ -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces , fungi such as yeasts, and animal cells, such as CHO, R1J, B-W and L-M cells, African Green Monkey kidney cells ⁇ e.g. , COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells ⁇ e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces , fungi such as yeasts
  • animal cells such as CHO, R1J, B-W and L-M cells, African Green Monkey kidney cells ⁇ e.g. , COS 1, COS 7, BSC1, B
  • the DNA encoding the sulfotransferase will be transfected into a suitable host along with the DNA encoding the neural cell adhesion molecule upon which the sulfotransferase acts. Further, the necessary glucuronyltransferase may be similarly expressed. The co-expression of the enzymes with the neural cell adhesion molecule thus results in the neural cell adhesion molecule then being transformed in situ with the desired HNK-1 carbohydrate epitope, thus enhancing its therapeutic capabilities.
  • the DNA or a gene encoding the sulfotransferase enzyme of the invention, or a protein or polypeptide domain fragment thereof is introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue- specific promoter, or both.
  • Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retro viral vectors. Methods for constructing and using viral vectors are known in the art [see, e.g. , Miller and Rosman, BioTechniques 7:980-990 (1992)].
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, adipose tissue can be specifically targeted.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell.
  • an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g. , adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells.
  • the viral vector e.g. , adenovirus vector
  • immunosuppressive cytokines such as interleukin-12 (IL-12), interferon- ⁇ (IFN- ⁇ ), or anti-CD4 antibody
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors [see, e.g. , Wilson, Nature Medicine (1995)].
  • a viral vector that is engineered to express a minimal number of antigens.
  • the DNA or gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al. , U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Patent No. 4,650,764; Temin et al. , U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62: 1120; Temin et al. , U.S. Patent No. 5,124,263; International Patent Publication No.
  • Retroviral vectors can be constructed to function as infections particles or to undergo a single round of transfection. In the former case, the virus is modified to retain all of its genes except for those responsible for oncogenic transformation properties, and to express the heterologous gene. Non-infectious viral vectors are prepared to destroy the viral packaging signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals. Thus, the viral particles that are produced are not capable of producing additional virus.
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker
  • liposomes Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, e.g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • Targeted peptides e.g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g. , Wu et al., 7. Biol. Chem. 267:963-967 (1992); Wu and Wu, 7. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc. Natl.
  • a gene therapy vector as described above employs a transcription control sequence that comprises the DNA consensus sequence recognized by the sulfotransferase enzyme of the invention, i.e. , an HNK-1 binding site, operably associated with a therapeutic heterologous gene inserted in the vector. That is, a specific expression vector of the invention can be used in gene therapy.
  • the sulfotranferase protein will be used, in vitro, to carry out the final step (sulfate addition) in an otherwise chemical synthesis of the HNK-1 carbohydrate.
  • the completed HNK-1 carbohydrate can then be coupled to any protein molecule for further use.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • sulfotransferase analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for examination of sulfotransferase material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of sulfotransferase coding sequences. Analogs exhibiting "sulfotransferase activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding sulfotransferase can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the sulfotransferase amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al , Science, 223: 1299 (1984); Jay et al., J. Biol. Chem. , 259:6311 (1984).
  • DNA sequences allow convenient construction of genes which will express sulfotransferase analogs or "muteins" .
  • DNA encoding muteins can be made by site-directed mutagenesis of native sulfotransferase genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present sulfotransferase.
  • the sulfotransferase can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular sulfotransferase activity in suspect target cells.
  • antibody (ies) to the sulfotransferase can be produced and isolated by standard methods including the well known hybridoma techniques.
  • the antibody (ies) to the sulfotransferase will be referred to herein as Ab. and antibody(ies) raised in another species as Abj.
  • sulfotransferase in cells can be ascertained by the usual immunological procedures applicable to such determinations.
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the sulfotransferase labeled with a detectable label, antibody Aty labeled with a detectable label, or antibody Ab 2 labeled with a detectable label.
  • the sulfotransferase forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • At ⁇ a characteristic property of At ⁇ is that it will react with Ab, .
  • a raised in one mammalian species has been used in another species as an antigen to raise the antibody Abj.
  • Ab 2 may be raised in goats using rabbit antibodies as antigens.
  • Abj therefore would be anti-rabbit antibody raised in goats.
  • Ab x will be referred to as a primary or anti-sulfotransferase antibody
  • Ab ⁇ will be referred to as a secondary or anti-A antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the sulfotransferase or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, B-D-glucosidase, B-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled sulfotransferase may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined sulfotransferase, and cell samples would then be inoculated and thereafter incubated.
  • the resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5 % .
  • Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay useful and contemplated in accordance with the present invention is known as a "cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose res obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined sulfotransferase activity or predetermined sulfotransferase activity capability in suspected target cells.
  • one class of such kits will contain at least the labeled sulfotransferase or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g. , "competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence or capability of cells for predetermined sulfotransferase activity, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol ⁇ e.g., “competitive, “ “sandwich, “ “double antibody, “ etc.), and comprises:
  • an assay system for screening potential drugs effective to modulate the activity of the sulfotransferase may be prepared.
  • the sulfotransferase may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the sulfotransferase activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known sulfotransferase.
  • Gal ⁇ -pNP 4-nitrophenyl- ⁇ -D-galactose
  • GlcA ⁇ l-3Gal ⁇ -R 2-heptanoylamidoethyl-(3-O- ⁇ -D-glucuronyl)- ⁇ -D-galactose;
  • GlcA ⁇ -pNP 4-nitrophenyl- ⁇ -D-glucuronic acid
  • PAPS 3'-phosphoadenosine-5'-phosphosulfate.
  • CHOP2 cells (Cummings et al, (1993) Biochem. Biophys. Res. Commun. 195:814- 822) were grown in MEM ⁇ medium supplemented with 10% fetal calf serum, penicillin/streptomycin and 200 ⁇ g/ml G418 (all from Life Technologies, Basel, Switzerland). For transfections G418 was omitted.
  • Hybridoma supernatants containing antibodies HNK-1 from mouse (Abo and Balch, (1981) 7. Immunology 127:1024-1029) and L2-412 from rat (Kruse et al., (1984) Nature 311:153-155) were produced as described (Noronha et al., (1986) Brain Res.
  • the glucuronyltransferase cDNA was in the mammalian expression vector pEF-BOS (Terayama et al, (1997) Proc. Natl. Acad. Sci., U.S.A. 94:6093-6098).
  • RNA from cerebral cortex of newborn rats was converted to double- stranded cDNA using a kit from Stratagene, and a 1-3 kb fraction prepared by gel electrophoresis was cloned into the vector pXMDl (Kluxen et al, (1992) Proc. Nat. Acad. Sci. U.S.A. 89:4618-4622). Plates with transformed colonies were replicated to nitrocellulose filters, which were then further processed for storage as filter "sandwiches" at -80°C (Hanahan and Meselson (1980) Gene 10:63-67). Bacteria remaining on the plates were regrown overnight and collected for preparation of plasmid DNA. Subpools were prepared by cutting replica sandwiches into 10-12 pieces and regrowing the bacteria from one side of the sandwich.
  • the sulfotransferase cDNA downstream from the Msc I site at nucleotide 319 was subcloned into the filled EcoRl site of plasmid pPROTA (Sanchez-Lopez et al., (1988) J. Biol. Chem. 263:11892-11899).
  • CHOP2 cells were grown in MEM ⁇ Medium (Life Technologies Cat. No. 11900- 016) supplemented with 10% fetal calf serum, Penicillin/Streptomycin and 200 ⁇ g/1 G418 (all from Life Technologies, Basel, Switzerland). During transfections medium without G418 was used. In cotransfection experiments 2 parts of test plasmid(s) were supplemented with 1 part plasmid coding for glucuronyltransferase (Terayama et al., (1997) Proc.Natl. Acad. Sci., U.S.A. 94:6093-6098). Cells were transfected using DEAE-dextran (Kluxen and L ⁇ bbert (1993.) Anal. Biochem.
  • CHOP2 cells transfected with either the sulfotransferase cDNA, glucuronyltransferase cDNA, or a control vector containing LacZ were scraped from the plate in PBS, pelleted, and resuspended in SDS-PAGE loading buffer. After boiling for 5 min. and recentrifugation the supernatant was subjected to standard 10% SDS-PAGE (Sambrook). An equivalent of 10 ⁇ g of protein was used per lane.
  • the Western blot was incubated with monoclonal antibody HNK-1 or L2- 412 as described for immunostaining of cells, except that skim milk powder (Fluka, Buchs, Switzerland) was used for blocking.
  • the sulfotransferase cDNA plasmid or a control plasmid containing the lacZ gene was transfected into CHOP2 cells as described for the cDNA pools on a proportionally larger scale.
  • the CHOP2 cells were harvested by scraping in PBS two days after transfection with the sulfotransferase cDNA plasmid. After centrifugation the cells from an 80 cm 2 flask were taken up in 250 ⁇ l of 100 mM bis-TRIS (pH 6.6) containing mixed protease inhibitors (chymostatin/ pepstatinA/ leupeptin/ antipain apoprotinin, all at 10 ⁇ g/ml). Aliquots were stored at -20°C and only thawed once. Cerebral cortex of 7-day-old rats was homogenized in the same buffer (25 % wt/vol) and similarly aliquoted.
  • the sulfotransferase assays were done in 100 mM BIS-TRIS (pH 6.6), 10 mM MnCl 2 , 2.5 mM ATP, and 0.1 % Triton X-100, in a final volume of 20 ⁇ l including 10 ⁇ l of the transfected cell homogenate (50 ⁇ g protein) or 5 ⁇ l of 25 % brain homogenate (30 ⁇ g protein), 100 pmol of [ 35 S]-PAPS (900 Bq; from New England Nuclear, diluted with unlabeled PAPS from Sigma), and 10 nmol of acceptor substrate.
  • acceptors used were 4-nitrophenyl- ⁇ -D-galactose (Gal ⁇ -pNP), 4- nitrophenyl- ⁇ -D-glucuronic acid (GlcA ⁇ -pNP) (both Fluka) or 2- heptanoylamidoethyl-(3-O- ⁇ -D-glucuronyl)- ⁇ -D-galactose (GlcA ⁇ l ⁇ 3Gal ⁇ -R; A.V. Kornilov, L.O. Kononov, A.A. Sherman, and N. E.
  • the remaining 35 S in the pellet (about 3%) is counted by liquid scintillation to subtract from the total amount in the assay.
  • the dried supernatant is redissolved in 10 ⁇ l 1: 1 water :methanol and loaded on aluminum supported HPTLC plates (silica gel 60, Merck) and run in 5:4: 1 Chloroform :methanol: 0.25% KCl/water.
  • Developed plates are layered overnight on a Phosphor Imager screen (Molecular Dynamics) and the activity is quantified by measuring the fraction of counts in the spots compared to the total lane. In FIGURE 3, a 7 day exposure of an X-ray film is shown.
  • EXAMPLE 6 Sequencing of the sulfotransferase cDNA The sulfotransferase cDNA clone was subcloned using Sall/Xhol in the Xhol site of pBluescriptKS (Stratagene). Several smaller subclones were made using internal BamHl, EcoRl, EcoRl, Mscl, Pstl and Smal sites. These fragments were sequenced using the universal Ml 3 and Ml 3 reversed primer. To cover the whole clone in two orientations, several sequence specific primers were used.
  • a Mscl/EcoRV fragment from the cDNA clone in pBluescriptKS was isolated by agarose electrophoresis and ligated into pPROTA digested with EcoRl and blunted using Klenow enzyme.
  • the sulfotransferase part of the encoded fusion protein starts with Serine 22, located at the end of the putative transmembrane domain.
  • HNK-1 sulfotransferase For the expression cloning of the HNK-1 sulfotransferase, a cDNA library was made from PO rat cerebral cortex, which at this age shows high expression of both the HNK-1 epitope (Chou et al., (1991) J. Neurochem. 57:852-859) and the enzymes synthesizing it (Chou and Jungalwala (1993) 7. Biol. Chem. 268:330-336). As a recipient cell line, the CHOP2 cell line was used (Cummings et al., (1993) Biochem. Biophys. Res. Commun. 195:814-822).
  • CHOP 2 is a derivative of the Lec2 cell line (Stanley and Siminovitch (1977) Somatic Cell Genet. 3:391-404), and lacks the CMP-sialic acid transporter; this mutation results in an increase in glycoproteins and glycolipids terminating in B4-Galactose.
  • the glucuronyltransferase acts on B4-Galactose residues (Chou et al. , (1991) J. Neurochem. 57:852-859), elimination of the sialytransferase activity may increase the amount of substrate available to the glucuronyl and sulfotransferases.
  • the system is, in principle, suitable for the cloning of a glucuronyltransferase as well as a sulfotransferase.
  • a glycoprotein-specific glucuronyltransferase was recently cloned after purification of the enzyme (Terayama et al, (1997) Proc.Natl Acad. Sci., U.S.A. 94:6093-6098).
  • the rat cerebral cortex cDNA library was divided into 40 pools of 5-10,000 clones. Plasmid DNA from cDNA pools was cotransfected with the glucuronyltransferase. Two days after transfection, the plates were screened for HNK-1 binding cells by immuno-staining. The number of colored cells in each plate was scored by systematically scanning the plate under a light microscope. In pool 3, about 20 cells were seen that reacted with the HNK-1 antibody, indicating the sulfotransferase was expressed in these cells (FIGURE 1A). Other pools showed only rare colored cells, and these looked generally "less healthy" than those seen in the positive pool.
  • HNK-1 reactivity after transfection with the sulfotransferase cDNA is dependent on the presence of the glucuronyltransferase. Only a very faint HNK-1 reactivity, but clearly higher than in mock transfected cells, is seen after transfection with the sulfotransferase alone (FIGURE IF).
  • sulfotransferase activity was confirmed by enzyme assays in vitro. Sulfotransferase activity was determined with Gal ⁇ -pNP, GlcA ⁇ -pNP and GlcA ⁇ l ⁇ 3Gal ⁇ -R as acceptors (FIGURE 3). Homogenates of cells transfected with the isolated sulfotransferase clone showed activity towards GlcA ⁇ -pNP and
  • GlcA ⁇ l ⁇ 3Gal ⁇ l-R but not to Gal ⁇ -pNP.
  • Homogenates of mock-transfected cells gave no sulfotransferase activity; cerebral cortex homogenate could use both GlcA ⁇ -pNP and Gal ⁇ -pNP as acceptor, suggesting the presence of more than one sulfotransferase in this tissue.
  • the sulfotransferase activity towards the disaccharide acceptor GlcA ⁇ l ⁇ 3Gal ⁇ -R in brain as measured from lane 4, panel A is 154 pmol/mg-h, very close to the maximal activity measured by Chou and Jungalwala (22) using a glycolipid acceptor.
  • Transfected CHOP2 cells show an activity of 80 pmol/mg-h. There was no increase in activity with longer oligosaccharides, and activity towards GlcA ⁇ -pNP was about 5 times lower in both rat brain and CHOP2 cells.
  • the enzyme encoded by the cloned cDNA is therefore able to perform the same transfer of sulfate to terminal ⁇ -linked GlcA residues as measured in rat cerebral cortex.
  • Soluble Ll/HNK-1 was made in CHOP2 cells as a recombinant Ll-Fc fusion protein with the HNK-1 carbohydrate using a procedure similar to that described in Neuron 14:57-66, 1995.
  • the later was the construct of LI in fusion with Fc as described in the above reference.
  • the recombinant protein was purified by Protein A affinity chromatography and used for subsequent studies.
  • the presence of the HNK-1 epitope was confirmed by positive reaction with HNK-1 -specific antibodies (Abo and Balch (1981) J. Immunology 127: 1024-1029).
  • Recombinant Ll/HNK-1 is functionally active and is a potent agent in neuronal survival
  • Recombinant HNK-1 glycosylated Ll-Fc, as prepared in Example 11, and non- glycosylated Ll-Fc were used as substrates coated onto plastic or as a soluble molecule added to the culture medium at approximately 1-10 ⁇ g/ml.
  • Neurite outgrowth and survival of mesencephalic neurons from day 17 rat embryos were examined in culture after 7 days in vitro maintenance.
  • Dopaminergic neurons were recognized by immunostaining for dopamine- ⁇ -hydroxylase (DBH) and quantified using IBAS morphometric equipment.
  • DBH dopamine- ⁇ -hydroxylase

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Abstract

L'isolement d'un ADNc de sulfotransférase via une stratégie de clonage d'expression a révélé une sulfotransférase comportant 356 acides aminés, avec des caractéristiques d'une protéine transmembranaire de type II. Lorsqu'elle est exprimée en tant que protéine hybride soluble, l'enzyme est capable de transférer le sulfate d'un donneur de sulfate vers des substrats accepteurs contenant de l'acide glucuronique terminal. La sulfotransférase isolée peut être utilisée pour préparer des protéines comportant l'épitope de carbohydrate HNK-1 qui est exprimé sur plusieurs glycoprotéines d'adhérence neurale et en tant que glycolipide, et participe dans les interactions cellulaires. La glucuronyltransférase et la sulfotransférase sont considérés comme étant des enzymes clés dans la biosynthèse de cet épitope, parce que le reste de la structure survient souvent dans des glycoconjugués. Par conséquent, l'invention concerne une méthode de sulfonation de l'acide glucuronique, présent dans des molécules d'adhérence cellulaire et d'adhérence cellulaire neurale, en exposant la molécule d'adhérence cellulaire neurale à une enzyme sulfotransférase recombinée pour générer, sur celle-ci, un épitope de carbohydrate HNK-1. L'utilisation d'ADNc isolé ou recombiné pour générer la protéine hybride L1-Fc recombinée glycosylée HNK-1 peut, dès lors, faciliter diverses méthodes thérapeutiques dans lesquelles on peut utiliser L1, ou d'autres membres de sa superfamille d'immunoglobulines, en particulier dans le traitement de neurones endommagés ou malades. La présente invention concerne également des méthodes de préparation de la sulfotransférase, de molécules d'adhérence neurale définissant, sur celle-ci, l'épitope carbohydrate HNK-1, des méthodes permettant de favoriser la croissance neurale et/ou la remyélinisation et/ou la neuroprotection, et des équipements diagnostiques, le tout utilisant les molécules et les matériels de l'invention.
PCT/US1998/018572 1997-09-05 1998-09-04 Sulfotransferase hnk-1 et ses methodes d'utilisation WO1999011796A1 (fr)

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EP1043333A4 (fr) * 1997-12-26 2001-11-28 Ono Pharmaceutical Co NOUVEAUX POLYPEPTIDES, ADNc CODANT CES POLYPEPTIDES ET UTILISATION DE CEUX-CI
WO2001063293A3 (fr) * 2000-02-24 2002-04-11 Oxford Glycosciences Uk Ltd Proteines et genes et leur utilisation dans le diagnostic et le traitement de la schizophrenie
US6441131B1 (en) 1997-12-22 2002-08-27 Ono Pharmaceutical Co., Ltd. Peptides, method for assaying human pepsinogen II or human pepsin II, and assaying kit

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AMJAD A. ILYAS ET AL.: "Variability in the structural requirements for binding of human monoclonal anti-myelin-associated glycoprotein immunoglobulin <<<m antibodies and HNK.1 to sphingoglycolipid antigens", JOURNAL OF NEUROCHEMISTRY, vol. 55, no. 2, August 1990 (1990-08-01), pages 594 - 601, XP002087868 *
BAKKER H ET AL: "Expression cloning of a cDNA encoding a sulfotransferase involved in the biosynthesis of the HNK -1 carbohydrate epitope.", JOURNAL OF BIOLOGICAL CHEMISTRY, (1997 NOV 21) 272 (47) 29942-6. JOURNAL CODE: HIV. ISSN: 0021-9258., United States, XP002087870 *
Emhum1 Database Entry Af033827 Accession number AF033827; 10 March 1998 ONG E. ET AL.:"Homo sapiens HNK-1 sulfotransferase mRNA, complete cds." *
KOICHI HONKE ET AL.: "Purification and characterization of 3'-Phosphoadenosine-5'-Phosphosulfate:GalCer Sulfotransferase from human renal cancer cells", THE JOURNAL OF BIOCHEMISTRY, vol. 119, no. 3, March 1996 (1996-03-01), pages 421 - 426, XP002087869 *
KOJI TERAYAMA ET AL.: "Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 94, no. 12, 10 June 1997 (1997-06-10), WASHINGTON US, pages 6093 - 6098, XP002087867 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6441131B1 (en) 1997-12-22 2002-08-27 Ono Pharmaceutical Co., Ltd. Peptides, method for assaying human pepsinogen II or human pepsin II, and assaying kit
EP1043333A4 (fr) * 1997-12-26 2001-11-28 Ono Pharmaceutical Co NOUVEAUX POLYPEPTIDES, ADNc CODANT CES POLYPEPTIDES ET UTILISATION DE CEUX-CI
WO2001063293A3 (fr) * 2000-02-24 2002-04-11 Oxford Glycosciences Uk Ltd Proteines et genes et leur utilisation dans le diagnostic et le traitement de la schizophrenie

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