WO2010082622A1 - NOVEL HIGH-FUNCTION ENZYME OBTAINED BY ALTERING SUBSTRATE SPECIFICITY OF HUMAN β-HEXOSAMINIDASE Β - Google Patents

NOVEL HIGH-FUNCTION ENZYME OBTAINED BY ALTERING SUBSTRATE SPECIFICITY OF HUMAN β-HEXOSAMINIDASE Β Download PDF

Info

Publication number
WO2010082622A1
WO2010082622A1 PCT/JP2010/050397 JP2010050397W WO2010082622A1 WO 2010082622 A1 WO2010082622 A1 WO 2010082622A1 JP 2010050397 W JP2010050397 W JP 2010050397W WO 2010082622 A1 WO2010082622 A1 WO 2010082622A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
subunit
protein
hexosaminidase
amino acids
Prior art date
Application number
PCT/JP2010/050397
Other languages
French (fr)
Japanese (ja)
Inventor
均 櫻庭
伊藤　孝司
大輔 辻
Original Assignee
学校法人明治薬科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人明治薬科大学 filed Critical 学校法人明治薬科大学
Priority to JP2010546654A priority Critical patent/JP5678664B2/en
Publication of WO2010082622A1 publication Critical patent/WO2010082622A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a recombinant protein having an activity derived from the ⁇ -subunit of wild-type human ⁇ -hexosaminidase.
  • Tay-Sachs disease and Sandhoff disease are both diseases in which GM2 ganglioside accumulates in nervous system cells due to decreased activity of ⁇ -hexosaminidase A (Hex A), resulting in cranial nerve symptoms.
  • Hex A is a heterodimer composed of an ⁇ -subunit and a ⁇ -subunit, and has an enzyme activity that degrades GM2 ganglioside.
  • Tay-Sachs disease is Hex A deficiency based on ⁇ -subunit deficiency
  • Sandhoff disease is Hex A deficiency based on ⁇ -subunit deficiency.
  • Fabry disease ⁇ -galactosidase deficiency
  • Pompe disease acid ⁇ -glucosidase deficiency
  • Gaucher disease glucocerebrosidase deficiency
  • mucopolysaccharidosis type I ⁇ -L- For the treatment of iduronidase deficiency
  • mucopolysaccharidosis type II idulsulfatase deficiency
  • mucopolysaccharidosis type VI allylsulfatase B deficiency
  • Chinese hamster ovary-derived cultured cells CHO cells
  • cultured human fibers Enzyme replacement therapy, in which a recombinant enzyme (deficient enzyme) produced in blast cells is administered intravascularly, has been developed and used in the clinical stage, and has been effective.
  • Wild-type human ⁇ -hexosaminidase A (Hex A; known to have an activity of degrading GM2 ganglioside consisting of a heterodimer of ⁇ -subunit and ⁇ -subunit) and human ⁇ -Regarding the production of hexosaminidase B (Hex B; a homodimer of ⁇ -subunit and known to have no activity of degrading GM2 ganglioside), cultured cells derived from baby hamster kidney ( BHK cells) are known (see Patent Document 1).
  • the present inventors also introduced an expression vector into which a gene encoding an ⁇ -subunit and a ⁇ -subunit (HEXA cDNA and HEXB cDNA, respectively) was inserted into a CHO cell line or a special yeast strain.
  • a gene encoding an ⁇ -subunit and a ⁇ -subunit HEXA cDNA and HEXB cDNA, respectively
  • GM2 ganglioside accumulated in the cranial nervous system was reduced and neurological symptoms were improved. The effectiveness of enzyme replacement therapy was observed.
  • the problem to be solved by the present invention is that, as an enzyme that can be used for enzyme replacement therapy for Tay-Sachs disease and Sandhoff disease, there are few adverse side effects such as allergic reaction and anaphylactic reaction, and blood (plasma) stability is low.
  • the object is to provide an enzyme protein and a gene encoding the protein which are high and are easily taken into cells of a damaged organ.
  • the present inventor has intensively studied to solve the above problems. As a result, by converting a part of the amino acid sequence of ⁇ -subunit into another specific amino acid residue based on the three-dimensional structure information of ⁇ -subunit and ⁇ -subunit of ⁇ -hexosaminidase A modified ⁇ -subunit having a substrate recognition function of ⁇ -subunit was prepared. Then, the present inventor produced a modified ⁇ -hexosaminidase B (modified Hex B) that is a homodimer with the modified ⁇ -subunit as a constituent component, and has an activity of degrading GM2 ganglioside. I found out.
  • modified Hex B modified Hex B
  • amino acids 312 to 315, 452 and 453 are replaced with another amino acid.
  • a protein having activity derived from the ⁇ -subunit of wild-type human ⁇ -hexosaminidase is replaced with another amino acid.
  • the protein of (2) above is, for example, a protein in which the 312st to 315th amino acids are sequentially replaced by glycine, serine, glutamic acid and proline, or a protein in which the 452nd amino acid is replaced by asparagine.
  • Examples include proteins in which an amino acid is substituted with asparagine and the 453rd amino acid is substituted with arginine.
  • a protein comprising any one of the following amino acid sequences (i) to (iv): (i) An amino acid in which the 312th to 315th amino acids in the amino acid sequence shown in SEQ ID NO: 4 are sequentially replaced with an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine.
  • the protein of the above (3) is, for example, a protein in which an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine are glycine, serine, glutamic acid and proline, respectively, or the aspartic acid A protein whose amino acid is other than asparagine, and a protein whose amino acid other than leucine is arginine.
  • an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine respectively.
  • Glycine, serine, glutamic acid and proline, and an amino acid other than the aspartic acid is asparagine
  • an amino acid other than the leucine is arginine.
  • a protein comprising a homodimer of the protein of any one of (1) to (3) above.
  • Base sequence (ii) Base sequence in which the 1354th to 1356th bases in the base sequence shown in SEQ ID NO: 3 are replaced with bases indicating codons of amino acids other than aspartic acid (iii) Base shown in SEQ ID NO: 3 A base sequence in which the 1357th to 1359th bases in the sequence are replaced with bases indicating codons of amino acids other than leucine (iv) in the base sequence shown in SEQ ID NO: 3, The 34th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases, in order, have codons for amino acids other than arginine, respectively.
  • the gene of (6) is, for example, a gene in which amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine and amino acids other than lysine are glycine, serine, glutamic acid, and proline, respectively, and the aspartic acid A gene whose amino acid is other than asparagine, and a gene whose amino acid other than leucine is arginine, and amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine, and amino acids other than lysine, respectively.
  • Glycine, serine, glutamic acid and proline, an amino acid other than the aspartic acid is asparagine
  • an amino acid other than the leucine is arginine.
  • the 312th to 315th amino acids are respectively replaced with glycine, serine, glutamic acid and proline, the 452nd amino acid is replaced with asparagine, and the 453rd amino acid is replaced with arginine. Can be done.
  • a pharmaceutical composition for treating Tay-Sachs disease comprising the protein according to any one of (1) to (4) above.
  • a pharmaceutical composition for treating Tay-Sachs disease comprising the gene of (5) or (6) above.
  • a method for treating Tay-Sachs disease comprising administering the pharmaceutical composition of (12) and / or the pharmaceutical composition of (13) to a Tay-Sachs disease patient.
  • a pharmaceutical composition for treating Sandhoff's disease comprising the protein according to any one of (1) to (4) above.
  • a pharmaceutical composition for treating Sandhoff disease comprising the gene of (5) or (6) above.
  • a method for treating Tay-Sachs disease comprising administering the pharmaceutical composition of (15) and / or the pharmaceutical composition of (16) to a Tay-Sachs disease patient.
  • an enzyme that can be used for enzyme replacement therapy for Tay-Sachs disease and Sandhoff disease there are few adverse side effects such as allergic reaction and anaphylactic reaction, high blood (plasma) stability,
  • An enzyme protein and a gene encoding the protein that can be easily taken up by cells can be provided.
  • the protein of the present invention and the gene encoding the protein are extremely useful in that they can be used for the creation of excellent novel high-functional enzyme therapeutic agents for Tay-Sachs disease and Sandhoff disease, which are Hex A deficiencies.
  • FIG. 3 is a schematic diagram showing a three-dimensional structure of human ⁇ -hexosaminidase A (a heterodimer of ⁇ -subunit and ⁇ -subunit). It is a schematic diagram showing a functional region of the ⁇ -subunit of human ⁇ -hexosaminidase A. The diagram on the left shows the active pocket in the ⁇ -subunit and the GM2 activating protein binding site, and the diagram on the right shows the dimerization surface in the ⁇ -subunit.
  • FIG. 3 is a schematic diagram showing an aspect of amino acid substitution in the ⁇ -subunit necessary for giving the ⁇ -subunit the activity of degrading GM2 ganglioside.
  • This modified Hex B has a ⁇ -subunit but not an ⁇ -subunit because most of its molecular structure (especially the outer shell) is equal to that of the ⁇ -subunit. Even if administered, allergic reactions are unlikely to occur. Tay-Sachs disease is a genetic disease with a very high incidence, particularly in Jews (about 1 in 3,000 to 5,000 people), so if a therapeutic drug with few side effects is developed, its clinical effect will be extremely large.
  • HexB is more stable than Hex A
  • modified Hex B is also expected to be more stable than wild-type Hex A, and a sustained effect can be expected.
  • HexB has the advantage that it has a larger number of sugar chains than Hex A.
  • HexA and Hex B are taken up by cells such as the nervous system via a mannose 6-phosphate receptor on the cell membrane. Therefore, the greater the number of mannose 6-phosphate residues at the sugar chain end of the enzyme protein, the higher the efficiency of incorporation into cells. If the number of sugar chains of modified HexB is greater than that of wild-type Hex A, it is expected that the efficiency of incorporation into nervous system cells is high, and the clinical effect is also considered to be high. In this respect, it can be said that the modified HexB has an advantage not only as a therapeutic agent for Tay-Sachs disease but also as a therapeutic agent for Sandhoff disease.
  • the part corresponding to these specific amino acid residues in the ⁇ -subunit molecule (the 452nd amino acid residue, the 453rd amino acid residue, the 312th to 315th amino acids) Amino acid substitution was performed in the order of “D452N”, “L453R”, and “RQNK (312-315) GSEP”, respectively.
  • the notation of “D452N” is an embodiment in which the amino acid residue at position 452 of the ⁇ -subunit is substituted from aspartic acid to asparagine, and the notation of “L453R” is the position at position 453 of the ⁇ -subunit.
  • the amino acid residue is substituted from leucine to arginine
  • the notation “RQNK (312-315) GSEP” represents four consecutive amino acid residues from the 312th position to the 315th position of the ⁇ -subunit,
  • arginine is replaced with glycine
  • glutamine is replaced with serine
  • asparagine is replaced with glutamic acid
  • lysine is replaced with proline.
  • the protein of the present invention comprises a mutant protein of the ⁇ -subunit of wild-type human ⁇ -hexosaminidase and a modified (mutant) enzyme (specifically, a dimer of the mutant protein).
  • Human ⁇ -hexosaminidase B modified Hex B
  • the protein of the present invention changes the structure of the active site (especially the substrate binding site) of the ⁇ -subunit and is necessary for the association (binding) with the GM2 activator ( ⁇ -subunit).
  • the structural change described above is not limited to a structural change that completely disables the binding of the ⁇ -subunit to the substrate, and is inherently more than the binding reactivity of the ⁇ -subunit to the substrate. It also includes a structural change that allows the binding reactivity of the unit with the substrate to be relatively high, while conversely, the binding reactivity of the ⁇ -subunit with the substrate becomes significantly high.
  • ⁇ -subunit substrate specificity means the structure of the active site (particularly, the position and type of amino acid residues that play an important role in substrate binding reactivity) and the GM2 activator. It means that the presence of the loop structure necessary for the association (binding) is the same as that of the ⁇ -subunit.
  • Examples of the protein of the present invention include, for example, at least one amino acid among amino acids 312 to 315, 452 and 453 in the amino acid sequence of ⁇ -subunit.
  • An amino acid sequence substituted with another amino acid (more preferably, an amino acid sequence in which amino acids at positions 312 to 315, 452 and 453 are all substituted with other amino acids), or the above-mentioned substituted
  • a protein comprising an amino acid sequence in which one or several amino acids except amino acids 312 to 315, 452 and 453 are deleted, substituted or added, and ⁇ - Preferred is a protein having an activity derived from a subunit.
  • deletion, substitution or addition is preferably performed in a portion excluding the signal peptide of the ⁇ -subunit.
  • the signal peptide is a portion consisting of the first to 54th amino acids in the amino acid sequence of the ⁇ -subunit.
  • GenBank accession number: NM 000512 "and” Accession number: NM "000521” and is registered as "Entry name: HEXB-HUMAN; Accession number: P07686” in Swiss-Prot (available from http://tw.expasy.org/uniprot/).
  • GenBank information on the amino acid sequence of the ⁇ -subunit (SEQ ID NO: 2) and the base sequence encoding the sequence (SEQ ID NO: 1) are also included in GenBank, for example, “Accession number: NM 000511 "and” Accession number: NM “000520” and registered in Swiss-Prot (available from http://tw.expasy.org/uniprot/) as "Entry name: HEXA-HUMAN; Accession number: P06865".
  • the base sequence (cDNA) encoding the amino acid sequence of the ⁇ -subunit shown in SEQ ID NO: 1 is GenBank (Accession number: NM 000520) is a base sequence composed of the 208th to 1797th bases in the base sequence of 2437 bp in total.
  • the nucleotide sequence (cDNA) encoding the amino acid sequence of the ⁇ -subunit shown in SEQ ID NO: 3 is GenBank (Accession number: NM 000521) is a base sequence composed of the 118th to 1788th bases in a base sequence of 1919 bp in total.
  • the “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is, for example, about 1 to 10, preferably about 1 to 5 amino acids deleted or substituted. Or, it is preferably an added amino acid sequence.
  • amino acids other than lysine (Lys: K) but are not particularly limited.
  • glycine, serine, glutamic acid, and proline are preferably mentioned in this order.
  • the 452nd amino acid residue is not particularly limited as long as it is other than aspartic acid (Asp: D), and for example, asparagine (Asn: N) is preferably exemplified.
  • the amino acid residue at position 453 is not particularly limited as long as it is other than leucine (Leu: L), and preferred examples include arginine (Arg: R).
  • the 312th to 315th amino acids are glycine, serine, glutamic acid and proline, respectively, and the 452nd amino acid is asparagine, and the 453rd amino acid. Is particularly preferably arginine.
  • the amino acid after the substitution does not substantially affect the structure composed of other unsubstituted amino acids.
  • the effect of allowing recognition of an acidic substrate can be obtained by substituting the 452nd and 453rd amino acids present in the substrate binding site, respectively, as described above. Further, by substituting each of the 312st to 315th amino acids as described above, it is possible to obtain an effect that a loop structure necessary for association (binding) with the GM2 activator can be introduced.
  • the GM2 activator acts on the trap between the enzyme protein and the substrate (GM2 ganglioside) and is an important factor for exerting the activity derived from the ⁇ -subunit.
  • the protein of the present invention is also preferably the following protein (a) or (b).
  • sequence (ii) An amino acid sequence in which the 452nd amino acid is substituted with an amino acid other than aspartic acid in the amino acid sequence shown in SEQ ID NO: 4
  • the 453rd amino acid is other than leucine (Iv) in the amino acid sequence shown in SEQ ID NO: 4
  • the 312th to 315th amino acids are respectively an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and Substituted with an amino acid other than lysine and the 452nd position Amino acid is replaced by an amino acid other than aspartic acid, and the amino acid sequence # 453 amino acid has been substituted with an amino acid other than leucine
  • amino acid sequence of any one of (i) to (iv) above comprising an amino acid sequence in which one or several amino acids except the amino acid at the substitution site are deleted, substituted or added, and wild-type human ⁇ -Protein having activity derived from ⁇ -subunit of hexosaminidase
  • amino acids other than arginine, amino acids other than glutamine, other than asparagine in the description of (i) and (iv) above Preferred examples include proteins in which “amino acids other than lysine” are glycine, serine, glutamic acid, and proline, respectively.
  • a protein in which the ⁇ amino acid other than aspartic acid '' in the description of (ii) and (iv) above is asparagine, or the protein in the description of (iii) and (iv) above.
  • a protein in which the “amino acid other than leucine” is arginine is also preferred.
  • the protein (a) is more preferably a protein comprising the amino acid sequence (iv) among the proteins comprising the amino acid sequences (i) to (iv), and comprises the amino acid sequence represented by SEQ ID NO: 6.
  • a protein is more preferred, and a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is particularly preferred.
  • the ⁇ -subunit-derived activity is confirmed by, for example, collecting and expressing the target protein in cells derived from mammals such as CHO cells and human fibroblasts, and measuring 4-MUGS degradation activity. it can.
  • the protein (enzyme solution) and 4-methylumbelliferyl-N-acetyl- ⁇ -D-glucosamine-6-sulfate (4-methylumbelliferyl-6-sulfo-N-acetyl- ⁇ -D- 4-methylumbelliferone that can be released per unit time when the enzyme solution is mixed with glucosaminide (artificial substrate) and reacted under pH 4.5 conditions.
  • glucosaminide artificial substrate
  • reacted under pH 4.5 conditions can be measured by detecting the amount of.
  • various known detection methods can be adopted. For example, a detection method using a fluorometer or the like is preferable.
  • the target protein may be expressed by incorporating it into various known expression vectors and introducing it into cells.
  • the gene encoding the protein of the present invention described above is not limited, but a gene containing the following DNA (a) or (b) is preferably exemplified.
  • the following DNAs (a) and (b) are preferably both structural genes of the protein of the present invention, but a gene containing these DNAs may be composed only of these DNAs.
  • these DNAs may be included in part, and may also include known base sequences (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression, and are not limited.
  • Base sequence (ii) Base sequence in which the 1354th to 1356th bases in the base sequence shown in SEQ ID NO: 3 are replaced with bases indicating codons of amino acids other than aspartic acid (iii) Base shown in SEQ ID NO: 3 A base sequence in which the 1357th to 1359th bases in the sequence are replaced with bases indicating codons of amino acids other than leucine (iv) in the base sequence shown in SEQ ID NO: 3, The 34th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases, in order, have codons for amino acids other than arginine, respectively.
  • the term “codon” means not only a three-base chain (triplet) on the RNA sequence after transcription but also a three-base chain on the DNA sequence. Therefore, the codon notation on the DNA sequence is performed using thymine (T) instead of uracil (U).
  • the base sequence shown in SEQ ID NO: 3 is a base sequence consisting of 1671 bases encoding the ⁇ -subunit (556 amino acids) of wild-type human ⁇ -hexosaminidase.
  • a base indicating a codon of an amino acid other than arginine, a base indicating a codon of an amino acid other than glutamine, a codon of an amino acid other than asparagine is a base indicating a codon of glycine, a base indicating a codon of serine, a base indicating a codon of glutamic acid, and a base indicating a codon of proline, respectively.
  • DNA is preferred.
  • DNA that indicates the codon of the amino acid other than aspartic acid '' is a base that indicates the codon of asparagine
  • Preferable examples also include DNAs in which the “bases indicating the codons of amino acids other than leucine” in the descriptions of (iii) and (iv) above are bases indicating the codons of arginine.
  • the bases indicating the codons of the above amino acids are “ggt”, “ggc”, “gga”, or “ggg”. (Preferably “ggg”) and the base indicating the codon of serine is “tct”, “tcc”, “tca”, “tcg”, “agt” or “agc” (preferably “tct”),
  • the base indicating the codon for glutamic acid is “gag” or “gaa” (preferably “gag”)
  • the base indicating the codon for proline is “cct”, “ccc”, “cca” or “ccg” (preferably “ ccc ")
  • the base indicating the codon of asparagine is "but as an amino acid other than the above aspartic acid) is” aat “or” aac "(preferably” aac ")
  • the base indicating the codon of arginine is “Cgt
  • the DNA of the above (a) is more preferably a DNA containing the base sequence of the above (iv) among the DNAs containing the base sequences of the above (i) to (iv), including the base sequence shown in SEQ ID NO: 5.
  • DNA is more preferred, and DNA consisting of the base sequence shown in SEQ ID NO: 5 is particularly preferred.
  • Such mutation-substituted DNA is described in, for example, Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. It can be prepared according to the site-specific displacement induction method. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, and examples of the kit include QuickChange TM Site.
  • PCR primers designed to introduce a missense mutation so that a base representing the codon of the desired amino acid is introduced using a DNA containing a base sequence encoding the ⁇ -subunit as a template under appropriate conditions It can also be prepared by PCR.
  • the DNA polymerase used for PCR is not limited, it is preferably a highly accurate DNA polymerase.
  • Pwo DNA Polymerase Roche Diagnostics
  • Pfu DNA polymerase Promega
  • Platinum Pfx DNA polymerase Invitrogen
  • KOD DNA polymerase Toyobo
  • KOD-plus-polymerase Toyobo
  • the PCR reaction conditions may be appropriately set depending on the optimum temperature of the DNA polymerase to be used, the length and type of DNA to be synthesized, etc.
  • cycle conditions “90 to 98 ° C. for 5 to 30 seconds (thermal denaturation / Dissociation) ⁇ 50 to 65 ° C. for 5 to 30 seconds (annealing) ⁇ 65 to 80 ° C. for 30 to 1200 seconds (synthesis / elongation) ”is preferably performed under a total of 20 to 200 cycles.
  • the DNA of (b) is a DNA comprising any of the nucleotide sequences (i) to (iv) (that is, the DNA of (a) above), a DNA comprising a complementary nucleotide sequence, or a fragment thereof.
  • the obtained product can be used as a probe and subjected to known hybridization methods such as colony hybridization, plaque hybridization, and Southern blot, and can be obtained from a cDNA library or a genomic library.
  • a library prepared by a known method may be used, or a commercially available cDNA library or genomic library may be used, and is not limited.
  • Molecular Cloning A Laboratory Manual 2nd ed.
  • “Stringent conditions” in carrying out the hybridization method are conditions at the time of washing after hybridization, wherein the buffer salt concentration is 15 to 330 mM, the temperature is 25 to 65 ° C., preferably the salt concentration is 15 to It means the condition of 150mM and temperature of 45 ⁇ 55 °C. Specifically, for example, conditions such as 50 mM at 80 mM can be mentioned. Furthermore, in addition to such conditions as salt concentration and temperature, various conditions such as probe concentration, probe length, reaction time, etc. are also considered, and conditions for obtaining the DNA of (b) above should be set as appropriate. Can do.
  • the hybridizing DNA is preferably a base sequence having at least 40% homology with the DNA base sequence of (a) above, more preferably 60%, even more preferably 90% or more, particularly Preferably it is 95% or more, Most preferably, it is 99% or more.
  • the base corresponding to the base at the substitution site is the same as the base at the substitution site.
  • the “corresponding base” of the “base corresponding to the base of the substitution site” means that when the DNA of (b) is hybridized with a complementary strand to the DNA of (a), It means a base (triplet) which is in a position opposite to the base complementary to the base at the substitution site (triplet).
  • the base sequence of the DNA of (b) is free of deletion and addition mutations compared to the DNA of (a) (that is, the length (number of bases) of both DNAs is the same).
  • the 934th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases are the “corresponding bases”.
  • the DNA of (b) is preferably one in which the base sequence region encoding the signal peptide of the ⁇ -subunit is the same as the DNA of (a).
  • the base sequence region encoding the signal peptide is a region composed of the first to 162nd bases in the base sequence shown in SEQ ID NO: 3.
  • the base sequence is not completely the same as the DNA of (a) above but the amino acid sequence after translation is completely the same.
  • a DNA consisting of a sequence that is, a DNA obtained by subjecting the DNA of (a) to a silent mutation
  • codons corresponding to individual amino acids after translation are not particularly limited.
  • codons generally used in mammals such as humans after transcription preferably frequency of use
  • DNA that shows a codon that is commonly used in microorganisms such as E. coli and yeast, plants, etc. (preferably a codon that is frequently used). May be included.
  • Recombinant Vector and Transformant In order to express the protein of the present invention, it is first necessary to construct a recombinant vector by incorporating the above-described gene of the present invention into an expression vector. At this time, a transcription promoter, SD sequence (when the host is a prokaryotic cell) and Kozak sequence (when the host is a eukaryotic cell) are ligated upstream in advance as necessary to the gene to be incorporated into the expression vector. Alternatively, a terminator may be linked downstream, and an enhancer, splicing signal, poly A addition signal, selection marker, etc. may be linked. Each element necessary for gene expression such as the above transcription promoter may be included in the gene from the beginning, or may be used when originally included in the expression vector.
  • a use aspect is not specifically limited.
  • various methods using known gene recombination techniques such as a method using a restriction enzyme and a method using topoisomerase can be employed.
  • the expression vector is not limited as long as it can hold the gene encoding the protein of the present invention, such as plasmid DNA, bacteriophage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, and the like.
  • a vector suitable for the host cell to be used can be appropriately selected and used.
  • the constructed recombinant vector is introduced into a host to obtain a transformant, which is cultured, whereby the protein of the present invention can be expressed.
  • the “transformant” as used in the present invention means a gene into which a foreign gene has been introduced into the host, for example, a gene into which a foreign gene has been introduced by introducing plasmid DNA or the like into the host (transformation), Also included are those in which a foreign gene has been introduced by infecting a host with various viruses and phages (transduction).
  • the host is not limited as long as it can express the protein of the present invention after the introduction of the above recombinant vector, and can be selected as appropriate. For example, various animal cells such as humans and mice can be selected.
  • hosts such as various plant cells, bacteria, yeast, and plant cells.
  • animal cells for example, human fibroblasts, CHO cells, baby hamster kidney-derived cultured cells (BHK cells) monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells, etc. Used.
  • Insect cells such as Sf9 cells and Sf21 cells can also be used.
  • the codon type of the gene contained in the recombinant vector may be the same as or different from the codon type of the host actually used, and is not limited.
  • the protein of the present invention comprises: The structure of the active site (especially the substrate binding site) of the wild-type human ⁇ -hexosaminidase ⁇ -subunit is changed so that the substrate of the wild-type human ⁇ -hexosaminidase ⁇ -subunit can bind. And a structure capable of binding to the GM2 ganglioside activator (loop structure; structure existing in the ⁇ -subunit).
  • a structural change can be performed, for example, by substituting a specific amino acid residue in the amino acid sequence constituting the ⁇ -subunit with another amino acid residue, as described above, by a genetic recombination technique or the like. it can.
  • the production of the protein of the present invention is carried out by a method including the step of culturing the above-described transformant and the step of collecting a protein having activity derived from ⁇ -subunit from the obtained culture.
  • “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells.
  • the transformant can be cultured according to a usual method used for host culture.
  • the protein of interest is accumulated in the culture.
  • any known natural medium and any known medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host, and can efficiently culture the transformant.
  • Any synthetic medium may be used.
  • the cells may be cultured under selective pressure in order to prevent the recombinant vector contained in the transformant from dropping and the gene encoding the target protein from dropping off. That is, when the selectable marker is a drug resistance gene, the corresponding drug can be added to the medium, and when the selectable marker is an auxotrophic complementary gene, the corresponding nutrient factor can be removed from the medium. it can.
  • G418 when culturing human fibroblasts transduced with a vector containing a G418 resistance gene, G418 (G418 sulfate) may be added as needed during the culture.
  • a suitable inducer eg, IPTG
  • the culture conditions of the transformant are not particularly limited as long as the productivity of the target protein and the growth of the host are not hindered, and are usually 10 ° C to 40 ° C, preferably 20 ° C to 37 ° C, and 5 to 100. Do time.
  • the pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.
  • the target protein When the target protein is produced in the microbial cells or cells after culturing, the target protein can be collected by disrupting the microbial cells or cells.
  • the cells or cell crushing residues can be removed as necessary.
  • the method for removing the residue include centrifugation and filtration. If necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid.
  • the supernatant obtained after removing the residue is a cell extract soluble fraction and can be a crudely purified protein solution.
  • the target protein when the target protein is produced in the microbial cells or cells, the microbial cells and the cells themselves can be recovered by centrifugation, membrane separation, etc., and used without being crushed.
  • the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration. Then, if necessary, the target protein is collected from the culture by extraction with ammonium sulfate precipitation, and further, if necessary, using dialysis and various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.) It can also be isolated and purified.
  • the production yield of the protein obtained by culturing the transformant is, for example, SDS-PAGE (polyacrylamide gel) in units such as per culture solution, per microbial wet weight or dry weight, or per crude enzyme solution protein. For example, electrophoresis).
  • the target protein can also be produced using a cell-free protein synthesis system that does not use any living cells.
  • the cell-free protein synthesis system is a system that synthesizes a target protein in an artificial container such as a test tube using a cell extract.
  • Cell-free protein synthesis systems that can be used also include cell-free transcription systems that synthesize RNA using DNA as a template.
  • the cell extract to be used is preferably derived from the aforementioned host cell.
  • cell extracts include extracts derived from eukaryotic cells or prokaryotic cells. More specifically, CHO cells, rabbit reticulocytes, mouse L-cells, HeLa cells, wheat germ, budding yeast, Escherichia coli, and the like are extracted. Liquid can be used. These cell extracts may be used after being concentrated or diluted, or may be used as they are, and are not limited.
  • the cell extract can be obtained, for example, by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. Such cell-free protein synthesis can also be performed using a commercially available kit.
  • PEG polyethylene glycol
  • reagent kits PROTEIOS TM (Toyobo), TNT TM System (Promega), synthesizer PG-Mate TM (Toyobo), RTS (Roche Diagnostics) and the like.
  • the target protein produced by cell-free protein synthesis can be purified by appropriately selecting means such as chromatography as described above.
  • composition i) Pharmaceutical composition as a replenishment enzyme, etc.
  • the protein of the present invention can exert various excellent effects on the treatment of Tay-Sachs disease and Sandhoff disease. It can be used as an active ingredient of a therapeutic agent for Tay-Sachs disease and a therapeutic agent for Sandhoff disease. That is, the present invention provides a pharmaceutical composition for the treatment of Tay-Sachs disease (Tea-Sachs disease therapeutic agent) and a pharmaceutical composition for the treatment of Sandhoff disease (A therapeutic agent for Sandhoff disease) containing the protein of the present invention described above. It is. Specifically, these pharmaceutical compositions are preferably enzyme enzymes for replenishment that can be used for enzyme replacement therapy.
  • the protein of the present invention used in these pharmaceutical compositions is particularly preferably a homodimer (ie, modified Hex B).
  • the protein of the present invention which is an active ingredient in the pharmaceutical composition, may be used in the state of various salts, hydrates, etc., if necessary, and also has storage stability as a therapeutic agent (particularly activity maintenance). ) May be used in a state in which appropriate chemical modification is made, and is not limited.
  • the pharmaceutical composition can contain other components besides the protein of the present invention.
  • the other components include various pharmaceutical components (various pharmaceutically acceptable carriers and the like) required depending on the usage (form of use) of the pharmaceutical composition.
  • Other components can be appropriately contained as long as the effects exhibited by the protein of the present invention are not impaired.
  • the proportion of the protein of the present invention and the types and proportions of other components should be set appropriately according to known methods for preparing replenishment enzymes. Can do.
  • the administration of the pharmaceutical composition is not limited, but in the case of a supplementary enzyme, parenteral methods such as intravenous infusion are usually employed.
  • preparations that can be used for various methods include excipients, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, and buffering agents that are commonly used in pharmaceutical production.
  • Preservatives, solubilizers, preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and used, and can be prepared by conventional methods.
  • the form of the pharmaceutical composition is not limited, but in the case of a replenishing enzyme drug, an intravenous injection (including infusion) is usually adopted, for example, a unit dose ampoule or a multi-dose container state Etc. may be provided.
  • the dosage of the pharmaceutical composition is generally determined in consideration of the compounding ratio of the active ingredient in the preparation, and the administration subject (patient) age, weight, type of disease, medical condition, administration route, number of administrations, A wide range can be set as appropriate in consideration of the administration period and the like.
  • the therapeutic agent of the present invention is a supplementary enzyme drug
  • the number of administrations is preferably about once every 2 to 4 weeks, and the dosage (/ once) is, for example, the active ingredient.
  • the amount of the protein or the like (recombinant enzyme) of the invention is preferably such that it can be administered at about 0.1 to 10 mg / kg, more preferably about 0.1 to 5 mg / kg, still more preferably 0.2 to 1 mg / kg relative to the body weight of the patient. Degree.
  • the protein of the present invention (recombinant enzyme) as an active ingredient is excellent in blood stability and has a high efficiency of uptake into cells of damaged organs.
  • an excellent enzyme supplementation effect can be obtained, and since there are very few adverse effects such as allergic side effects, the physical, mental and economic burden on the patient can be greatly reduced.
  • the gene of the present invention encodes the protein of the present invention that can exert various excellent effects on the treatment of Tay-Sachs disease and Sandhoff disease.
  • Tey-Sachs disease treatment pharmaceutical composition Tey-Sachs disease treatment (specifically gene therapy agent)
  • Zandhoff disease treatment pharmaceutical composition Zandhoff disease treatment agent (specifically gene therapy agent))
  • the method of administering the vector in which the nucleic acid was integrated other than the method of administering directly by injection is mentioned.
  • the vector examples include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, and lentivirus vectors. It can administer efficiently by using these viral vectors.
  • a commercially available gene transfer kit for example, product name: Adeno Express, manufactured by Clontech
  • Adeno Express manufactured by Clontech
  • the pharmaceutical composition (gene therapy drug)
  • the endoplasmic reticulum holding the gene of the present invention is introduced into a predetermined cell by the lipofection method.
  • the obtained cells are administered, for example, intravenously or intraarterially. It can also be administered locally to an organ affected by Fabry disease.
  • the pharmaceutical composition is administered to an adult, it is preferably about 0.1 ⁇ g / kg to 1000 mg / kg per day, more preferably about 1 ⁇ g / kg to 100 mg / kg relative to the weight of the patient. is there.
  • the present invention includes a method for treating Tay-Sachs disease and a method for treating Sandhoff disease, characterized by administering the above-described pharmaceutical composition to a Tay-Sachs disease patient or a Sandhoff disease patient.
  • the present invention also provides the use of the above pharmaceutical composition or the protein and / or gene of the present invention for the treatment of Tay-Sachs disease or Sandhoff disease, and the manufacture of a medicament for the treatment of Tay-Sachs disease or Sandhoff disease. This includes the use of the above pharmaceutical composition or the protein and / or gene of the present invention.
  • the pharmaceutical composition used in the therapeutic method of the present invention is a pharmaceutical composition containing the protein of the present invention (the above “6.
  • human ⁇ -hexosaminidase A is composed of an ⁇ -subunit and a ⁇ -subunit. It was considered that the site that recognizes GM2 ganglioside as a substrate and the binding site of GM2 activator that associates GM2 ganglioside with an enzyme are in the ⁇ -subunit.
  • amino acid residue notation indicates the type and position of the amino acid residue in the amino acid sequence of the ⁇ -subunit (SEQ ID NO: 2). For example, “R178” is indicated by SEQ ID NO: 2. 178th arginine residue of the amino acid sequence shown.
  • ⁇ Amino acid substitution of ⁇ -subunit required for degradation of GM2 ganglioside ⁇ Amino acid substitution of ⁇ -subunit required for degradation of GM2 ganglioside>
  • amino acid substitution that enables recognition of acidic substrates D452N, L453R
  • amino acid substitution that accompanies the introduction of the loop structure required for association with GM2 activator RQNK (312-315) GSEP
  • the amino acid residues constituting the functional region on the ⁇ -subunit obtained in Example 1 the corresponding amino acid residues on the ⁇ -subunit were identified. Differences in amino acid sequence and three-dimensional structure of ⁇ -subunit and ⁇ -subunit were compared.
  • the 452nd amino acid residue (D) in the ⁇ -subunit is substituted with an amino acid residue (N) corresponding to the amino acid residue in the ⁇ -subunit, and The 453rd amino acid residue (L) in the ⁇ -subunit was substituted with an amino acid residue (R) corresponding to the amino acid residue in the ⁇ -subunit.
  • the amino acid sequence (RQNK) from 312 to 315 in the ⁇ -subunit is changed to the amino acid sequence (GSEP) corresponding to the amino acid sequence in the ⁇ -subunit. Replaced.
  • the amino acid sequence of the modified ⁇ -subunit that has been amino acid substituted in this way is the amino acid sequence shown in SEQ ID NO: 6.
  • the cDNA of the wild-type human ⁇ -hexosaminidase ⁇ -subunit is inserted into the multicloning site of the mammalian expression vector pCXN 2 for recombinant expression.
  • vectors were constructed pCXN 2 / HEXB.
  • cDNA of pCXN 2 / HEXB vector of ⁇ - subunit cDNA modified ⁇ - subunits carried out base substitutions in the constructed a recombinant expression vector pCXN 2 / mut-HEXB.
  • the 452nd amino acid D (aspartic acid) is replaced with N (asparagine)
  • the 453rd amino acid L (leucine) is replaced with R (arginine)
  • the 312th to The base of the codon corresponding to each amino acid residue was substituted so that the 315th amino acid sequence RQNK (arginine-glutamine-asparagine-lysine) was replaced with GSEP (glycine-serine-glutamic acid-proline).
  • the codon “gat” corresponding to the amino acid D is replaced with “aac” corresponding to N
  • the codon “ttg” corresponding to the amino acid L is replaced with “cgt” corresponding to R
  • Four codons “aga caa aac aag” corresponding to the amino acid sequence RQNK were replaced with “ggg tct gag ccc” corresponding to GSEP.
  • the base sequence of the cDNA encoding the modified ⁇ -subunit thus base-substituted is the base sequence shown in SEQ ID NO: 5.
  • pCXN 2 / HEXB and pCXN 2 / mut-HEXB were each introduced into CHO cells, and drug-resistant cell populations that constantly express each gene in the presence of a neomycin derivative (G418 sulfate) were selected. Furthermore, a CHO clone cell line that highly expresses each gene was established using a limiting dilution method.
  • ⁇ Hex activity in CHO cell extract and culture supernatant The drug-resistant cell population (60 mm dish) obtained in Example 3 was cultured for 3 days in the presence of 10% fetal bovine serum, and a cell extract was prepared. The cell population was cultured in a serum-free medium for 3 days, and the culture supernatant was collected.
  • Fig. 5 shows the measurement results of 4-MUG degrading enzyme activity (shown in black in Fig. 5) and 4-MUGS degrading enzyme activity (shown in black diagonal lines in Fig. 5) in the cell extract and culture supernatant. It was.
  • 4-MUG means 4-methylumbelliferyl-N-acetyl- ⁇ -D-glucosaminide, which is used when measuring Hex B activity. It is used as an artificial substrate.
  • 4-MUGS means 4-methylumbelliferyl-6-sulfo-N-acetyl- ⁇ -D-glucosaminide However, it is used as an artificial substrate when measuring Hex A activity.
  • each Hex isozyme fraction used for supplementation to cultured fibroblasts Each Hex from CHO cell extracts expressing wild-type human ⁇ -hexosaminidase ⁇ -subunit and modified cDNA using an anion exchanger (DEAE) column equilibrated with 10 mM NaPiB (pH 6.0) Isoenzymes were isolated. By gradient elution using NaCl, CHO-derived Hex A was recovered in the 200 mM NaCl fraction, and human Hex B and modified Hex B were recovered in the flow-through fraction. These crudely purified fractions were buffer exchanged with 10 mM NaPiB (pH 6.0), and used for a supplementation experiment on patient-derived fibroblasts.
  • DEAE anion exchanger
  • ⁇ Decomposition effect of GM2 ganglioside by modified Hex B administration Wild-type human Hex A (4-MUGS degradation activity: 500 nmol / h), wild-type human Hex B (4-MUG degradation activity: 900 nmol / h) in the culture fluid of cultured fibroblasts from patients with Zandhoff disease Then, modified Hex B (4-MUGS degradation activity: 2,000 nmol / h) was added, the cells were fixed after culturing for 3 days, and immunofluorescent staining was performed using an anti-GM2 ganglioside antibody. The results are shown in FIG.
  • ⁇ Properties of modified Hex B> The ability to hydrolyze the substrate GM2 ganglioside when comparing ⁇ -hexosaminidase A (Hex A), which is an isozyme of ⁇ -hexosaminidase, with ⁇ -hexosaminidase B (Hex B) Has only Hex A, no HexB, and it is known that Hex B has significantly higher heat stability than both.
  • Hex A and Hex B are both glycoproteins, and the number of sugar chains is known to be 6 in Hex A and 8 in Hex B. Since the cellular uptake of the enzyme is defined by the number of mannose 6-phosphate present at the sugar chain end, it was considered that the larger number of sugar chains is more advantageous for the uptake.
  • the modified Hex B had GM2 ganglioside resolution because the binding site between the substrate recognition site and the GM2 activator was changed to Hex A-like.
  • the stability is similar to Hex B, and the stability at 37 ° C in 30% wild-type mouse plasma is also Hex A. It was higher than that.
  • the modified Hex B has the function of degrading GM2 ganglioside accumulated in the body of patients with Tay-Sachs disease and Sandhoff disease, and allergic reaction is caused to Tay-Sachs disease patients who frequently occur by its administration. It was found to be promising as a therapeutic agent for these diseases because it has a lower risk of occurrence, is more stable than wild-type Hex A, and excels in cellular uptake.
  • Sequence number 5 Recombinant DNA
  • SEQ ID NO: 6 recombinant protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Provided is an enzyme protein which is usable as an enzyme for enzyme replacement therapy for Tay-Sachs disease and Sandoffs disease, has little harmful side effects such as allergic reaction or anaphylactic reaction, is highly stable in blood (plasma) and can be easily incorporated into cells of an injured organ. Also provided are a gene encoding said protein and so on. An altered β-subunit protein obtained by changing the structure at the active site of the β-subunit of wild type human β-hexosaminidase and thus acquiring an activity originating in the α-subunit of wild type human β-hexosaminidase; and a homo dimer protein (altered β-hexosaminidase B) of the same.

Description

ヒトβ-ヘキソサミニダーゼＢの基質特異性を変換した新規高機能酵素A novel high-functional enzyme that converts the substrate specificity of human β-hexosaminidase B
　本発明は、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有する組換えタンパク質に関する。 The present invention relates to a recombinant protein having an activity derived from the α-subunit of wild-type human β-hexosaminidase.
　テイ－サックス病及びザンドホッフ病は、いずれも、β-ヘキソサミニダーゼＡ（Hex A）の活性低下により、神経系細胞にGM2ガングリオシドが蓄積して脳神経症状を来たす疾患である。Hex Aは、α-サブユニットとβ-サブユニットから構成されるヘテロ二量体であり、GM2ガングリオシドを分解する酵素活性を持つ。テイ－サックス病は、α-サブユニット欠損に基づくHex A欠損症であり、ザンドホッフ病は、β-サブユニット欠損に基づくHex A欠損症である。
　従来、テイ－サックス病及びザンドホッフ病に対して実際に臨床に使用可能な根本的治療法は確立されていない。一方、これらの疾患に類似した、ファブリー病（α-ガラクトシダーゼ欠損症）、ポンペ病（酸性α-グルコシダーゼ欠損症）、ゴーシェ病（グルコセレブロシダーゼ欠損症）、ムコ多糖症Ｉ型（α-L-イズロニダーゼ欠損症）、ムコ多糖症II型（イズルスルファターゼ欠損症）、ムコ多糖症VI型（アリルスルファターゼB欠損症）等の治療に関しては、チャイニーズハムスター卵巣由来の培養細胞（CHO細胞）や培養ヒト線維芽細胞で生産した組換え酵素（欠損酵素）を血管内投与する、酵素補充療法が開発されて臨床段階でも使用され、治療効果を上げている。 Tay-Sachs disease and Sandhoff disease are both diseases in which GM2 ganglioside accumulates in nervous system cells due to decreased activity of β-hexosaminidase A (Hex A), resulting in cranial nerve symptoms. Hex A is a heterodimer composed of an α-subunit and a β-subunit, and has an enzyme activity that degrades GM2 ganglioside. Tay-Sachs disease is Hex A deficiency based on α-subunit deficiency, and Sandhoff disease is Hex A deficiency based on β-subunit deficiency.
Conventionally, there has not been established a fundamental treatment method that can actually be used clinically for Tay-Sachs disease and Sandhoff disease. On the other hand, similar to these diseases, Fabry disease (α-galactosidase deficiency), Pompe disease (acid α-glucosidase deficiency), Gaucher disease (glucocerebrosidase deficiency), mucopolysaccharidosis type I (α-L- For the treatment of iduronidase deficiency), mucopolysaccharidosis type II (idulsulfatase deficiency), mucopolysaccharidosis type VI (allylsulfatase B deficiency), Chinese hamster ovary-derived cultured cells (CHO cells) and cultured human fibers Enzyme replacement therapy, in which a recombinant enzyme (deficient enzyme) produced in blast cells is administered intravascularly, has been developed and used in the clinical stage, and has been effective.
　しかしながら、これらの酵素補充療法では、もともと当該酵素を体内に持たない患者に対して酵素を繰り返し投与することから、当該酵素に対する抗体が産生され、アレルギー反応を主とする有害副反応が高頻度に見られ、臨床において大きな問題となっていた。また、これらの組換え酵素は、野生型酵素の性質を有し、安定性が低く、細胞内取り込みが必ずしも良いとは限らないことから、治療に際しては１～２週に１回の割合で頻回に投与する必要があり、神経系細胞等には取り込まれ難く臨床効果が乏しいという欠陥がある。
野生型のヒトβ-ヘキソサミニダーゼＡ（Hex A；α-サブユニットとβ-サブユニットとのヘテロ二量体からなりGM2ガングリオシドを分解する活性を持つことが知られている）及びヒトβ-ヘキソサミニダーゼＢ（Hex B；β-サブユニットのホモ二量体からなりGM2ガングリオシドを分解する活性を持たないことが知られている）の産生に関しては、ベイビーハムスター腎臓由来の培養細胞（BHK細胞）で生産する方法が知られている（特許文献１参照）。本発明者らも、CHO細胞株や特殊酵母株に対して、α-サブユニット及びβ-サブユニットをコードする遺伝子（各々HEXA cDNA及びHEXB cDNA）が挿入された発現ベクターを導入し、野生型の組換えHex Aを恒常発現する細胞株を樹立している（特許文献２参照）。この方法で生産した野生型組換えHex Aを、ザンドホッフ病モデルマウスに投与したところ、脳神経系に蓄積していたGM2ガングリオシドの減量や神経症状の改善が見られ、ザンドホッフ病やテイ－サックス病に対する酵素補充療法の有効性が認められていた。 However, in these enzyme replacement therapies, since the enzyme is repeatedly administered to patients who do not have the enzyme in the body, antibodies against the enzyme are produced, and adverse side reactions such as allergic reactions occur frequently. It was seen and became a big problem in the clinic. In addition, these recombinant enzymes have the properties of wild-type enzymes, have low stability, and do not always have good uptake into cells. Therefore, treatment is frequently performed once every 1-2 weeks. There is a defect that it is difficult to be taken up by nervous system cells and the clinical effect is poor.
Wild-type human β-hexosaminidase A (Hex A; known to have an activity of degrading GM2 ganglioside consisting of a heterodimer of α-subunit and β-subunit) and human β -Regarding the production of hexosaminidase B (Hex B; a homodimer of β-subunit and known to have no activity of degrading GM2 ganglioside), cultured cells derived from baby hamster kidney ( BHK cells) are known (see Patent Document 1). The present inventors also introduced an expression vector into which a gene encoding an α-subunit and a β-subunit (HEXA cDNA and HEXB cDNA, respectively) was inserted into a CHO cell line or a special yeast strain. Has established a cell line that constantly expresses the recombinant Hex A (see Patent Document 2). When wild-type recombinant Hex A produced by this method was administered to a model mouse for Zandhoff disease, GM2 ganglioside accumulated in the cranial nervous system was reduced and neurological symptoms were improved. The effectiveness of enzyme replacement therapy was observed.
国際公開第03/092612号パンフレットInternational Publication No. 03/092612 Pamphlet 特開2002-369692号公報JP 2002-369692 A
　しかしながら、ファブリー病等の他の疾患の患者や当該疾患のモデル動物に、欠損酵素を含む治療薬の投与を繰り返すと、多くの場合において治療薬中の酵素が体内で異物として認識され、抗体が産生されてしまい、その結果、アレルギー反応やアナフィラキシー反応等の有害副反応が高頻度に出現する。このことは、遺伝子治療的な方法で酵素を補充する場合においても同様である。よって、野生型組換えHex Aをそのままテイ－サックス病やザンドホッフ病患者に投与する場合には、上述した場合と同様に、有害副反応が生じる恐れがある。また、野生型組換えHex Aは、血中（血漿中）安定性が低く、障害臓器の細胞（神経系細胞）への取り込み効率が低い。
　そこで、本発明が解決しようとする課題は、テイ－サックス病やザンドホッフ病の酵素補充療法に用い得る酵素として、アレルギー反応やアナフィラキシー反応等の有害副作用が少なく、血中（血漿中）安定性が高く、しかも障害臓器の細胞に取り込まれやすい、酵素タンパク質及び当該タンパク質をコードする遺伝子等を提供することにある。 However, when a therapeutic drug containing a defective enzyme is repeatedly administered to patients with other diseases such as Fabry disease or model animals of the disease, in many cases, the enzyme in the therapeutic drug is recognized as a foreign substance in the body, and the antibody As a result, adverse side reactions such as allergic reactions and anaphylactic reactions frequently occur. The same applies to the case where the enzyme is supplemented by a gene therapy method. Therefore, when wild-type recombinant Hex A is directly administered to patients with Tay-Sachs disease or Sandhoff disease, an adverse side reaction may occur as in the case described above. Wild-type recombinant Hex A has low blood (plasma) stability and low uptake efficiency into cells (nervous cells) of damaged organs.
Therefore, the problem to be solved by the present invention is that, as an enzyme that can be used for enzyme replacement therapy for Tay-Sachs disease and Sandhoff disease, there are few adverse side effects such as allergic reaction and anaphylactic reaction, and blood (plasma) stability is low. The object is to provide an enzyme protein and a gene encoding the protein which are high and are easily taken into cells of a damaged organ.
　本発明者は、上記課題を解決するべく鋭意検討を行った。その結果、β-ヘキソサミニダーゼのα-サブユニット及びβ-サブユニットの立体構造情報に基づいて、β-サブユニットのアミノ酸配列の一部を他の特定のアミノ酸残基に変換することにより、α-サブユニットの基質認識機能を持つ改変β-サブユニットを作製した。そして、本発明者は、この改変β-サブユニットを構成成分として、ホモ二量体である改変β-ヘキソサミニダーゼB（改変Hex B）を作製したところ、GM2ガングリオシドを分解する活性を有することを見出した。この新規酵素（改変Hex B）は、α-サブユニットを含まないため、テイ－サックス病患者に投与しても有害な免疫反応を起こす可能性が少なく、また本来的にHex Bは、Hex Aに比べて安定性が高く、糖鎖の数も多いことから、細胞内への取り込みに関与する糖鎖末端のマンノース6-リン酸残基の含有率も高いと考えられる。従って、上記改変Hex Bを用いれば、Hex A欠損症であるテイ－サックス病やザンドホッフ病に対して優れた新規高機能酵素治療薬を創出できることを見出し、本発明を完成した。 The present inventor has intensively studied to solve the above problems. As a result, by converting a part of the amino acid sequence of β-subunit into another specific amino acid residue based on the three-dimensional structure information of α-subunit and β-subunit of β-hexosaminidase A modified β-subunit having a substrate recognition function of α-subunit was prepared. Then, the present inventor produced a modified β-hexosaminidase B (modified Hex B) that is a homodimer with the modified β-subunit as a constituent component, and has an activity of degrading GM2 ganglioside. I found out. Since this novel enzyme (modified Hex B) does not contain α-subunits, it is unlikely to cause an adverse immune response when administered to patients with Tay-Sachs disease. Therefore, it is considered that the content of the mannose 6-phosphate residue at the end of the sugar chain involved in cellular uptake is high. Therefore, it has been found that the use of the above modified Hex B can create a novel high-performance enzyme therapeutic agent excellent for Tay-Sachs disease and Sandhoff disease, which are Hex A deficiencies, and has completed the present invention.
　すなわち、本発明は以下の通りである。
　（１）野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を変化させて、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を獲得したタンパク質。
　上記(1)のタンパク質は、例えば、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニットの基質特異性を有するものが挙げられる。 That is, the present invention is as follows.
(1) A protein that has acquired the activity derived from the α-subunit of wild-type human β-hexosaminidase by changing the structure of the active site of the β-subunit of wild-type human β-hexosaminidase.
Examples of the protein (1) above include those having the substrate specificity of the α-subunit of wild-type human β-hexosaminidase.
　（２）野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列において第312番目～第315番目、第452番目及び第453番目のアミノ酸のうち少なくとも１つのアミノ酸が他のアミノ酸に置換されたアミノ酸配列、又は当該置換されたアミノ酸配列のうち第312番目～第315番目、第452番目及び第453番目のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有する、タンパク質。
　上記(2)のタンパク質は、例えば、第312番目～第315番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換されたタンパク質や、第452番目のアミノ酸がアスパラギンに置換されたタンパク質や、第453番目のアミノ酸がアルギニンに置換されたタンパク質が挙げられ、また、第312番目～第315番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換され、かつ第452番目のアミノ酸がアスパラギンに置換され、かつ第453番目のアミノ酸がアルギニンに置換されたタンパク質が挙げられる。 (2) In the amino acid sequence of the β-subunit of wild-type human β-hexosaminidase, at least one of amino acids 312 to 315, 452 and 453 is replaced with another amino acid. Amino acid sequence in which one or several amino acids except the 312st to 315th, 452nd and 453th amino acids are deleted, substituted or added among the substituted amino acid sequences And a protein having activity derived from the α-subunit of wild-type human β-hexosaminidase.
The protein of (2) above is, for example, a protein in which the 312st to 315th amino acids are sequentially replaced by glycine, serine, glutamic acid and proline, or a protein in which the 452nd amino acid is replaced by asparagine. And a protein in which the 453rd amino acid is substituted with arginine, and the 312th to 315th amino acids are substituted with glycine, serine, glutamic acid and proline, respectively, and the 452nd amino acid. Examples include proteins in which an amino acid is substituted with asparagine and the 453rd amino acid is substituted with arginine.
　（３）以下の(a)又は(b)のタンパク質。
　 (a) 下記(i)～(iv)のいずれかのアミノ酸配列を含むタンパク質。
　  (i) 配列番号４に示されるアミノ酸配列において第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換されたアミノ酸配列
　  (ii) 配列番号４に示されるアミノ酸配列において第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換されたアミノ酸配列
　  (iii) 配列番号４に示されるアミノ酸配列において第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列
　  (iv) 配列番号４に示されるアミノ酸配列において、第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換され、かつ第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換され、かつ第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列
　 (b) 上記(i)～(iv)のいずれかのアミノ酸配列において前記置換部位のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質 (3) The following protein (a) or (b).
(a) A protein comprising any one of the following amino acid sequences (i) to (iv):
(i) An amino acid in which the 312th to 315th amino acids in the amino acid sequence shown in SEQ ID NO: 4 are sequentially replaced with an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine. Sequence (ii) An amino acid sequence in which the 452nd amino acid is substituted with an amino acid other than aspartic acid in the amino acid sequence shown in SEQ ID NO: 4 (iii) In the amino acid sequence shown in SEQ ID NO: 4, the 453rd amino acid is other than leucine (Iv) in the amino acid sequence shown in SEQ ID NO: 4, the 312th to 315th amino acids are respectively an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and Substituted with an amino acid other than lysine and the 452nd position An amino acid sequence in which a mino acid is substituted with an amino acid other than aspartic acid and the 453rd amino acid is substituted with an amino acid other than leucine; (b) the substitution site in the amino acid sequence of any one of (i) to (iv) above; A protein comprising an amino acid sequence in which one or several amino acids except amino acids are deleted, substituted or added, and having an activity derived from the α-subunit of wild-type human β-hexosaminidase
　上記(3)のタンパク質は、例えば、前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであるタンパク質や、前記アスパラギン酸以外のアミノ酸がアスパラギンであるタンパク質や、前記ロイシン以外のアミノ酸がアルギニンであるタンパク質が挙げられ、また、前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであり、かつ前記アスパラギン酸以外のアミノ酸がアスパラギンであり、かつ前記ロイシン以外のアミノ酸がアルギニンであるタンパク質が挙げられる。 The protein of the above (3) is, for example, a protein in which an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine are glycine, serine, glutamic acid and proline, respectively, or the aspartic acid A protein whose amino acid is other than asparagine, and a protein whose amino acid other than leucine is arginine.In addition, an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine, respectively. , Glycine, serine, glutamic acid and proline, and an amino acid other than the aspartic acid is asparagine, and an amino acid other than the leucine is arginine.
　（４）上記(1)～(3)のいずれかのタンパク質のホモ二量体からなる、タンパク質。 (4) A protein comprising a homodimer of the protein of any one of (1) to (3) above.
　（５）上記(1)～(3)のいずれかのタンパク質をコードする遺伝子。 (5) A gene encoding any of the proteins (1) to (3) above.
　（６）以下の(a)又は(b)のDNAを含む遺伝子。
　 (a) 下記(i)～(iv)のいずれかの塩基配列を含むDNA
　  (i) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (ii) 配列番号３に示される塩基配列において第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (iii) 配列番号３に示される塩基配列において第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (iv) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換され、かつ第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換され、かつ第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　 (b) 上記(i)～(iv)のいずれかの塩基配列を含むDNAに対し相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、前記置換部位の塩基に対応する塩基が当該置換部位の塩基と同一であり、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質をコードするDNA (6) A gene containing the following DNA (a) or (b):
(a) DNA comprising any one of the nucleotide sequences (i) to (iv) below
(i) In the base sequence shown in SEQ ID NO: 3, the 934th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases Were sequentially replaced with a base indicating an amino acid codon other than arginine, a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, respectively. Base sequence (ii) Base sequence in which the 1354th to 1356th bases in the base sequence shown in SEQ ID NO: 3 are replaced with bases indicating codons of amino acids other than aspartic acid (iii) Base shown in SEQ ID NO: 3 A base sequence in which the 1357th to 1359th bases in the sequence are replaced with bases indicating codons of amino acids other than leucine (iv) in the base sequence shown in SEQ ID NO: 3, The 34th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases, in order, have codons for amino acids other than arginine, respectively. Is replaced with a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, and the 1354th to 1356th bases are other than aspartic acid. (B) any one of (i) to (iv) above, wherein the nucleotides 1357 to 1359 are replaced with bases indicating amino acid codons other than leucine. A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising any of the base sequences, wherein the base corresponding to the base of the substitution site is DNA encoding a protein that is identical to the base and has an activity derived from the α-subunit of wild-type human β-hexosaminidase
　上記(6)の遺伝子は、例えば、前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンである遺伝子や、前記アスパラギン酸以外のアミノ酸がアスパラギンである遺伝子や、前記ロイシン以外のアミノ酸がアルギニンである遺伝子が挙げられ、また、前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであり、かつ前記アスパラギン酸以外のアミノ酸がアスパラギンであり、かつ前記ロイシン以外のアミノ酸がアルギニンである遺伝子が挙げられる。 The gene of (6) is, for example, a gene in which amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine and amino acids other than lysine are glycine, serine, glutamic acid, and proline, respectively, and the aspartic acid A gene whose amino acid is other than asparagine, and a gene whose amino acid other than leucine is arginine, and amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine, and amino acids other than lysine, respectively. , Glycine, serine, glutamic acid and proline, an amino acid other than the aspartic acid is asparagine, and an amino acid other than the leucine is arginine.
　（７）上記(5)又は(6)の遺伝子を含む組換えベクター。 (7) A recombinant vector containing the gene of (5) or (6) above.
　（８）上記(7)の組換えベクターを含む形質転換体。 (8) A transformant comprising the recombinant vector of (7) above.
　（９）野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を変化させることにより、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を付与することを特徴とする、酵素の基質特異性変換方法。
　上記(9)の方法において、前記活性部位の構造の変化は、例えば、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列（具体的には、配列番号４に示されるアミノ酸配列）における第312番目～第315番目のアミノ酸を、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換し、かつ第452番目のアミノ酸をアスパラギンに置換し、かつ第453番目のアミノ酸をアルギニンに置換することにより行うことができる。 (9) By imparting an activity derived from the α-subunit of wild-type human β-hexosaminidase by changing the structure of the active site of the β-subunit of wild-type human β-hexosaminidase. A method for converting substrate specificity of an enzyme.
In the method of (9) above, the change in the structure of the active site is, for example, the amino acid sequence of the β-subunit of wild-type human β-hexosaminidase (specifically, the amino acid sequence represented by SEQ ID NO: 4). ), The 312th to 315th amino acids are respectively replaced with glycine, serine, glutamic acid and proline, the 452nd amino acid is replaced with asparagine, and the 453rd amino acid is replaced with arginine. Can be done.
　（10）野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニットの基質が結合できるように変化させることを特徴とする、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質の製造方法。 (10) It is characterized by changing the structure of the active site of the β-subunit of wild-type human β-hexosaminidase so that the substrate of the α-subunit of wild-type human β-hexosaminidase can be bound. A method for producing a protein having an activity derived from the α-subunit of wild-type human β-hexosaminidase.
　（11）上記(8)の形質転換体を培養する工程と、得られる培養物から野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質を採取する工程とを含む、当該タンパク質の製造方法。 (11) culturing the transformant of (8) above, and collecting a protein having an activity derived from the α-subunit of wild-type human β-hexosaminidase from the obtained culture, A method for producing the protein.
　（12）上記(1)～(4)のいずれかのタンパク質を含むことを特徴とする、テイ－サックス病治療用医薬組成物。 (12) A pharmaceutical composition for treating Tay-Sachs disease, comprising the protein according to any one of (1) to (4) above.
　（13）上記(5)又は(6)の遺伝子を含むことを特徴とする、テイ－サックス病治療用医薬組成物。 (13) A pharmaceutical composition for treating Tay-Sachs disease, comprising the gene of (5) or (6) above.
　（14）上記(12)の医薬組成物及び／又は上記(13)の医薬組成物をテイ－サックス病患者に投与することを特徴とする、テイ－サックス病の治療方法。 (14) A method for treating Tay-Sachs disease, comprising administering the pharmaceutical composition of (12) and / or the pharmaceutical composition of (13) to a Tay-Sachs disease patient.
　（15）上記(1)～(4)のいずれかのタンパク質を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。 (15) A pharmaceutical composition for treating Sandhoff's disease comprising the protein according to any one of (1) to (4) above.
　（16）上記(5)又は(6)の遺伝子を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。 (16) A pharmaceutical composition for treating Sandhoff disease, comprising the gene of (5) or (6) above.
　（17）上記(15)の医薬組成物及び／又は上記(16)の医薬組成物をテイ－サックス病患者に投与することを特徴とする、テイ－サックス病の治療方法。 (17) A method for treating Tay-Sachs disease, comprising administering the pharmaceutical composition of (15) and / or the pharmaceutical composition of (16) to a Tay-Sachs disease patient.
　本発明によれば、テイ－サックス病やザンドホッフ病の酵素補充療法に用い得る酵素として、アレルギー反応やアナフィラキシー反応等の有害副作用が少なく、血中（血漿中）安定性が高く、しかも障害臓器の細胞に取り込まれやすい、酵素タンパク質及び当該タンパク質をコードする遺伝子等を提供することができる。
　本発明のタンパク質及び当該タンパク質をコードする遺伝子は、Hex A欠損症であるテイ‐サックス病やザンドホッフ病に対する優れた新規高機能酵素治療薬の創出に利用できる点で極めて有用なものである。 According to the present invention, as an enzyme that can be used for enzyme replacement therapy for Tay-Sachs disease and Sandhoff disease, there are few adverse side effects such as allergic reaction and anaphylactic reaction, high blood (plasma) stability, An enzyme protein and a gene encoding the protein that can be easily taken up by cells can be provided.
The protein of the present invention and the gene encoding the protein are extremely useful in that they can be used for the creation of excellent novel high-functional enzyme therapeutic agents for Tay-Sachs disease and Sandhoff disease, which are Hex A deficiencies.
ヒトβ-ヘキソサミニダーゼA（α-サブユニットとβ-サブユニットとのヘテロ二量体）の三次元構造を示す模式図である。FIG. 3 is a schematic diagram showing a three-dimensional structure of human β-hexosaminidase A (a heterodimer of α-subunit and β-subunit). ヒトβ-ヘキソサミニダーゼAのα-サブユニットの機能領域を示す模式図である。左側の図は、α-サブユニット中の活性ポケット及びGM2活性化タンパク質結合部位を示し、右側の図は、α-サブユニット中の二量体形成面を示す。It is a schematic diagram showing a functional region of the α-subunit of human β-hexosaminidase A. The diagram on the left shows the active pocket in the α-subunit and the GM2 activating protein binding site, and the diagram on the right shows the dimerization surface in the α-subunit. β-サブユニットにGM2ガングリオシドの分解活性を持たせるために必要な、β-サブユニットにおけるアミノ酸置換の態様を示す模式図である。FIG. 3 is a schematic diagram showing an aspect of amino acid substitution in the β-subunit necessary for giving the β-subunit the activity of degrading GM2 ganglioside. 細胞株の樹立の手順（左）及び構築した発現ベクターの概略（右）を示す図である。It is a figure which shows the outline (right) of the procedure (left) of cell line establishment, and the constructed expression vector. CHO細胞抽出液及び培養上清におけるHex活性を示す図である。It is a figure which shows Hex activity in a CHO cell extract and a culture supernatant. 細胞抽出液及び培養上清における改変Hex B等の発現を示す図である。It is a figure which shows expression of modified Hex B etc. in a cell extract and a culture supernatant. 改変Hex B等の投与によるGM2ガングリオシドの分解効果を示す図である。It is a figure which shows the degradation effect of GM2 ganglioside by administration of modified Hex B.
　以下、本発明を詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。なお、本明細書は、本願優先権主張の基礎となる特願2009-008039号明細書（2009年1月16日出願）の全体を包含する。また、本明細書において引用された全ての刊行物、例えば先行技術文献、及び公開公報、特許公報その他の特許文献は、参照として本明細書に組み込まれる。 Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. In addition, this specification includes the whole of Japanese Patent Application No. 2009-008039 specification (filed on Jan. 16, 2009) which is the basis of the priority claim of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.
 
１．本発明の概要
　今回の発明では、ヒトβ-ヘキソサミニダーゼA（Hex A；図１参照）及びヒトβ-ヘキソサミニダーゼＢ（Hex B）のＸ線結晶構造情報に基づき、α-サブユニットとβ-サブユニットの立体構造を比較し、GM2ガングリオシドを分解するための基質認識やGM2活性化因子との相互作用に必要なアミノ酸残基を予測した。そして、in silicoで、いくつかのアミノ酸残基を置換することで、α-サブユニットとしての機能を持つ改変β-サブユニットを設計した。
　この設計に基づいてCHO細胞で産生された改変Hex Bは、野生型Hex Bが本来持たないGM2ガングリオシド分解能を獲得したものであった。この改変Hex Bは、その分子構造のほとんどが（特に外殻が）β-サブユニットのそれに等しいため、β-サブユニットは持っているが、α-サブユニットを持たないテイ－サックス病患者に投与しても、アレルギー反応を生じ難いものと考えられる。テイ－サックス病は、特にユダヤ人では極めて発症頻度が高い遺伝病であるため（約3,000～5,000人に1人）、副作用が少ない治療薬が開発されれば、その臨床効果は極めて大きい。また、Hex Aに比べてHexBは安定性が高いため、改変Hex Bも野生型Hex Aに比べて安定であることが予測され、効果の持続性が期待できる。さらに、HexBは、Hex Aに比べて糖鎖の数が多いという利点がある。HexAやHex Bは、神経系などの細胞に対しては細胞膜上のマンノース6-リン酸受容体を介して取り込まれることが知られている。そのため、酵素タンパク質の糖鎖末端のマンノース6-リン酸残基の数が多い程、細胞内への取り込み効率が高い。改変HexBの糖鎖数が野生型Hex Aよりも多いことは、神経系細胞への取り込み効率が高いことが予想され、臨床効果も高くなると考えられる。この点で、テイ－サックス病の治療薬のみならず、ザンドホッフ病の治療薬としても、改変HexBの優位性があると言える。
1. Summary of the Invention In the present invention, based on the X-ray crystal structure information of human β-hexosaminidase A (Hex A; see FIG. 1) and human β-hexosaminidase B (Hex B), By comparing the three-dimensional structure of the unit and β-subunit, we predicted the amino acid residues necessary for substrate recognition and interaction with GM2 activator to degrade GM2 ganglioside. Then, a modified β-subunit having a function as an α-subunit was designed by substituting several amino acid residues in silico.
The modified Hex B produced in CHO cells based on this design has acquired GM2 ganglioside resolution not inherent in wild type Hex B. This modified Hex B has a β-subunit but not an α-subunit because most of its molecular structure (especially the outer shell) is equal to that of the β-subunit. Even if administered, allergic reactions are unlikely to occur. Tay-Sachs disease is a genetic disease with a very high incidence, particularly in Jews (about 1 in 3,000 to 5,000 people), so if a therapeutic drug with few side effects is developed, its clinical effect will be extremely large. In addition, since HexB is more stable than Hex A, modified Hex B is also expected to be more stable than wild-type Hex A, and a sustained effect can be expected. Furthermore, HexB has the advantage that it has a larger number of sugar chains than Hex A. It is known that HexA and Hex B are taken up by cells such as the nervous system via a mannose 6-phosphate receptor on the cell membrane. Therefore, the greater the number of mannose 6-phosphate residues at the sugar chain end of the enzyme protein, the higher the efficiency of incorporation into cells. If the number of sugar chains of modified HexB is greater than that of wild-type Hex A, it is expected that the efficiency of incorporation into nervous system cells is high, and the clinical effect is also considered to be high. In this respect, it can be said that the modified HexB has an advantage not only as a therapeutic agent for Tay-Sachs disease but also as a therapeutic agent for Sandhoff disease.
　以下、本発明の概要を、(i)～(v)の手順を追って、より具体的に説明する。
　(i) ヒトHex A（α-サブユニットとβ-サブユニットのヘテロ二量体）とHex B（β-サブユニットのホモ二量体）のＸ線結晶構造情報に基づいて、α-サブユニット分子のうち、GM2ガングリオシドを基質として認識するための活性ポケット内のアミノ酸残基及びGM2活性化因子（当該酵素とその基質であるGM2ガングリオシドとの邂逅に働く）との結合に関係するアミノ酸残基を予測した。この予測を基に、β-サブユニット分子のうち、これらの特定アミノ酸残基に相当する部分（第452番目のアミノ酸残基、第453番目のアミノ酸残基、第312番目～第315番目のアミノ酸残基）を、それぞれ順に、「D452N」、「L453R」、「RQNK(312-315)GSEP」の態様でアミノ酸置換した。なお、「D452N」の表記は、β-サブユニットの第452番目のアミノ酸残基を、アスパラギン酸からアスパラギンに置換する態様であり、「L453R」の表記は、β-サブユニットの第453番目のアミノ酸残基を、ロイシンからアルギニンに置換する態様であり、「RQNK(312-315)GSEP」の表記は、β-サブユニットの第312番目～第315番目の４つの連続するアミノ酸残基を、それぞれ順に、アルギニンからグリシンに置換し、グルタミンからセリンに置換し、アスパラギンからグルタミン酸に置換し、リシンからプロリンに置換する態様を示す。 Hereinafter, the outline of the present invention will be described in more detail following the procedures (i) to (v).
(i) α-subunit based on X-ray crystal structure information of human Hex A (heterodimer of α-subunit and β-subunit) and Hex B (homodimer of β-subunit) Among the molecules, amino acid residues in the active pocket for recognizing GM2 ganglioside as a substrate and amino acid residues related to binding with GM2 activator (acting as a trap between the enzyme and its substrate, GM2 ganglioside) Predicted. Based on this prediction, the part corresponding to these specific amino acid residues in the β-subunit molecule (the 452nd amino acid residue, the 453rd amino acid residue, the 312th to 315th amino acids) Amino acid substitution was performed in the order of “D452N”, “L453R”, and “RQNK (312-315) GSEP”, respectively. The notation of “D452N” is an embodiment in which the amino acid residue at position 452 of the β-subunit is substituted from aspartic acid to asparagine, and the notation of “L453R” is the position at position 453 of the β-subunit. In this embodiment, the amino acid residue is substituted from leucine to arginine, and the notation “RQNK (312-315) GSEP” represents four consecutive amino acid residues from the 312th position to the 315th position of the β-subunit, In each case, arginine is replaced with glycine, glutamine is replaced with serine, asparagine is replaced with glutamic acid, and lysine is replaced with proline.
　(ii) β-サブユニットをコードするcDNA（HEXB cDNA）の発現ベクターを構築し、これに上記のアミノ酸置換が含まれるように、公知の遺伝子組換え技術を用いて変異を導入し、改変HEXB cDNAを作製した。
　(iii) この改変HEXB cDNAを、チャイニーズハムスター卵巣由来の培養細胞（CHO細胞）株に導入し、改変Hex Bを発現させ、その発現株を単離した。
　(iv) この細胞株の細胞抽出液及び分泌液中の改変Hex Bは、4-MUGS分解活性（通常用いられる、人工基質を用いた酵素活性測定方法で測定した場合のHex A活性を表す）を有した。また、この改変Hex Bをイムノブロット法で確認したところ、α-サブユニットに対する抗体に反応するタンパク質を含まないことが確認された。なお、上記「4-MUGS」は、4-メチルウムベリフェリル-N-アセチル-β-D-グルコサミン-6-硫酸（4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide）を意味し、Hex A活性測定の際に人工基質として用いられるものである。
　(v) この改変Hex Bを患者由来の培養線維芽細胞の培養液中に投与した後、免疫染色法で解析したところ、改変Hex Bが細胞内に取り込まれ、細胞内に蓄積していたGM2ガングリオシドの分解が認められた。
　なお、本明細書においては、単に「α-サブユニット」や「β-サブユニット」と表記した場合は、特に言及した場合を除き、野生型ヒトβ-ヘキソサミニダーゼ（Hex AやHex B）を構成する野生型α-サブユニット及び野生型β-サブユニットのことを意味する。 (ii) constructing an expression vector for cDNA encoding the β-subunit (HEXB cDNA), introducing a mutation using a known gene recombination technique so that the amino acid substitution described above is included therein, and modifying HEXB cDNA was prepared.
(iii) The modified HEXB cDNA was introduced into a cultured cell (CHO cell) line derived from Chinese hamster ovary to express the modified Hex B, and the expression strain was isolated.
(iv) Modified Hex B in the cell extract and secretion of this cell line is 4-MUGS degradation activity (represents Hex A activity as measured by a commonly used enzyme activity measurement method using an artificial substrate) Had. Further, when this modified Hex B was confirmed by immunoblotting, it was confirmed that it did not contain a protein that reacts with an antibody against the α-subunit. The above “4-MUGS” refers to 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulfate (4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide). This means that it is used as an artificial substrate when measuring Hex A activity.
(v) When this modified Hex B was administered into a culture fluid of patient-derived cultured fibroblasts and then analyzed by immunostaining, the modified Hex B was taken up into the cells and accumulated in the cells. Degradation of ganglioside was observed.
In the present specification, when simply expressed as “α-subunit” or “β-subunit”, wild-type human β-hexosaminidase (Hex A or Hex B, unless otherwise specified). ) Constituting the wild type α-subunit and the wild type β-subunit.
 
２．タンパク質
　本発明のタンパク質は、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの変異体タンパク質、及び当該変異体タンパク質の二量体からなる改変型（変異型）酵素（具体的には、ヒトβ-ヘキソサミニダーゼＢ（改変Hex B））である。
　詳しくは、本発明のタンパク質は、β-サブユニットの活性部位（特に基質結合部位）の構造を変化させ、かつ、GM2活性化因子との会合（結合）に必要なループ構造（α-サブユニットには存在する）を導入することにより、α-サブユニット由来の活性を獲得したタンパク質、及びその二量体タンパク質であり、好ましくは、当該α-サブユニットの基質特異性を有するタンパク質、及びその二量体タンパク質である。
　ここで、「α-サブユニット由来の活性を獲得した」とは、β-サブユニットの基質結合部位において、β-サブユニットの基質との結合反応性よりもα-サブユニットの基質との結合反応性が相対的に高くなったことを意味する。従って、上述した構造変化としては、β-サブユニットの基質との結合を完全に不可能にする構造変化には限定されず、本来α-サブユニットの基質との結合反応性よりもβ-サブユニットの基質との結合反応性が相対的に有意に高かったものを、逆にα-サブユニットの基質との結合反応性が有意に高くなるようにする構造変化も含む。また「α-サブユニットの基質特異性を有する」とは、活性部位の構造（特に、基質の結合反応性に重要な役割を果たすアミノ酸残基の位置及び種類）や、GM2活性化因子との会合（結合）に必要なループ構造の存在が、α-サブユニットと同様であることを意味する。
2. Protein The protein of the present invention comprises a mutant protein of the β-subunit of wild-type human β-hexosaminidase and a modified (mutant) enzyme (specifically, a dimer of the mutant protein). Human β-hexosaminidase B (modified Hex B)).
Specifically, the protein of the present invention changes the structure of the active site (especially the substrate binding site) of the β-subunit and is necessary for the association (binding) with the GM2 activator (α-subunit). A protein that has acquired activity derived from the α-subunit by introducing a dimer protein, preferably a protein having the substrate specificity of the α-subunit, and its It is a dimeric protein.
Here, “acquired activity derived from α-subunit” means that the binding of the α-subunit to the substrate rather than the binding reactivity of the β-subunit to the substrate at the substrate-binding site of the β-subunit. It means that the reactivity became relatively high. Therefore, the structural change described above is not limited to a structural change that completely disables the binding of the β-subunit to the substrate, and is inherently more than the binding reactivity of the α-subunit to the substrate. It also includes a structural change that allows the binding reactivity of the unit with the substrate to be relatively high, while conversely, the binding reactivity of the α-subunit with the substrate becomes significantly high. In addition, “having α-subunit substrate specificity” means the structure of the active site (particularly, the position and type of amino acid residues that play an important role in substrate binding reactivity) and the GM2 activator. It means that the presence of the loop structure necessary for the association (binding) is the same as that of the α-subunit.
　本発明のタンパク質（ホモ二量体も含む）としては、例えば、β-サブユニットのアミノ酸配列における第312番目～第315番目、第452番目及び第453番目のアミノ酸のうちの少なくとも１つのアミノ酸が他のアミノ酸に置換されたアミノ酸配列（より好ましくは、第312番目～第315番目、第452番目及び第453番目のアミノ酸がいずれも他のアミノ酸に置換されたアミノ酸配列）、又は前記置換されたアミノ酸配列のうち第312番目～第315番目、第452番目及び第453番目のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるタンパク質であって、かつα-サブユニット由来の活性を有するタンパク質が好ましく挙げられる。また、当該欠失、置換若しくは付加は、β-サブユニットのシグナルペプチドを除く部分においてなされることが好ましい。当該シグナルペプチドは、β-サブユニットのアミノ酸配列における第1番目～第54番目のアミノ酸からなる部分である。 Examples of the protein of the present invention (including homodimers) include, for example, at least one amino acid among amino acids 312 to 315, 452 and 453 in the amino acid sequence of β-subunit. An amino acid sequence substituted with another amino acid (more preferably, an amino acid sequence in which amino acids at positions 312 to 315, 452 and 453 are all substituted with other amino acids), or the above-mentioned substituted A protein comprising an amino acid sequence in which one or several amino acids except amino acids 312 to 315, 452 and 453 are deleted, substituted or added, and α- Preferred is a protein having an activity derived from a subunit. In addition, the deletion, substitution or addition is preferably performed in a portion excluding the signal peptide of the β-subunit. The signal peptide is a portion consisting of the first to 54th amino acids in the amino acid sequence of the β-subunit.
　なお、β-サブユニットのアミノ酸配列（配列番号４）及び当該配列をコードする塩基配列（配列番号３）の情報は、例えば、GenBankには「Accession number：NM 000512」及び「Accession number：NM 000521」として公表されており、Swiss-Prot（http://tw.expasy.org/uniprot/ から取得可能）には「Entry name：HEXB-HUMAN；Accession number：P07686」として登録されている。また、α-サブユニットのアミノ酸配列（配列番号２）及び当該配列をコードする塩基配列（配列番号１）の情報も同様に、例えば、GenBankには「Accession number：NM 000511」及び「Accession number：NM 000520」として公表されており、Swiss-Prot（http://tw.expasy.org/uniprot/ から取得可能）には「Entry name：HEXA-HUMAN；Accession number：P06865」として登録されている。ただし、配列番号１に示されるα-サブユニットのアミノ酸配列をコードする塩基配列（cDNA）は、GenBank（Accession number：NM 000520）に公表されている計2437 bpの塩基配列中の第208番目～1797番目の塩基からなる塩基配列である。同様に、配列番号３に示されるβ-サブユニットのアミノ酸配列をコードする塩基配列（cDNA）は、GenBank（Accession number：NM 000521）に公表されている合計1919 bpの塩基配列中の第118番目～1788番目の塩基からなる塩基配列である。
　ここで、上記「１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列」としては、例えば、１個～１０個程度、好ましくは１個～５個程度のアミノ酸が欠失、置換又は付加されたアミノ酸配列であることが好ましい。 The information on the amino acid sequence of the β-subunit (SEQ ID NO: 4) and the base sequence encoding the sequence (SEQ ID NO: 3) can be found in GenBank, for example, “Accession number: NM   000512 "and" Accession number: NM   "000521" and is registered as "Entry name: HEXB-HUMAN; Accession number: P07686" in Swiss-Prot (available from http://tw.expasy.org/uniprot/). Similarly, information on the amino acid sequence of the α-subunit (SEQ ID NO: 2) and the base sequence encoding the sequence (SEQ ID NO: 1) are also included in GenBank, for example, “Accession number: NM   000511 "and" Accession number: NM   "000520" and registered in Swiss-Prot (available from http://tw.expasy.org/uniprot/) as "Entry name: HEXA-HUMAN; Accession number: P06865". However, the base sequence (cDNA) encoding the amino acid sequence of the α-subunit shown in SEQ ID NO: 1 is GenBank (Accession number: NM   000520) is a base sequence composed of the 208th to 1797th bases in the base sequence of 2437 bp in total. Similarly, the nucleotide sequence (cDNA) encoding the amino acid sequence of the β-subunit shown in SEQ ID NO: 3 is GenBank (Accession number: NM   000521) is a base sequence composed of the 118th to 1788th bases in a base sequence of 1919 bp in total.
Here, the “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is, for example, about 1 to 10, preferably about 1 to 5 amino acids deleted or substituted. Or, it is preferably an added amino acid sequence.
　上述した他のアミノ酸としては、第312番目～第315番目のアミノ酸残基に関しては、それぞれ順に、アルギニン(Arg：R)以外のアミノ酸、グルタミン(Gln：Q)以外のアミノ酸、アスパラギン(Asn：N)以外のアミノ酸、及びリシン(Lys：K)以外のアミノ酸であれば特に限定はされないが、例えば、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンが好ましく挙げられる。同様に、第452番目のアミノ酸残基に関しては、アスパラギン酸(Asp：D)以外であれば特に限定はされないが、例えば、アスパラギン(Asn：N)が好ましく挙げられる。同様に、第453番目のアミノ酸残基に関しては、ロイシン(Leu：L)以外であれば特に限定はされないが、例えば、アルギニン(Arg：R)が好ましく挙げられる。中でも、上述した他のアミノ酸は、第312番目～第315番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであり、かつ第452番目のアミノ酸がアスパラギンであり、かつ第453番目のアミノ酸がアルギニンであることが特に好ましい。なお、上記置換後のアミノ酸は、他の置換されていないアミノ酸からなる構造に実質的に影響を及ぼさないものであることが好ましい。 As the other amino acids mentioned above, with respect to the 312th to 315th amino acid residues, amino acids other than arginine (Arg: R), amino acids other than glutamine (Gln: Q), and asparagine (Asn: N) in that order, respectively. ) And amino acids other than lysine (Lys: K), but are not particularly limited. For example, glycine, serine, glutamic acid, and proline are preferably mentioned in this order. Similarly, the 452nd amino acid residue is not particularly limited as long as it is other than aspartic acid (Asp: D), and for example, asparagine (Asn: N) is preferably exemplified. Similarly, the amino acid residue at position 453 is not particularly limited as long as it is other than leucine (Leu: L), and preferred examples include arginine (Arg: R). Among the above-mentioned other amino acids, the 312th to 315th amino acids are glycine, serine, glutamic acid and proline, respectively, and the 452nd amino acid is asparagine, and the 453rd amino acid. Is particularly preferably arginine. In addition, it is preferable that the amino acid after the substitution does not substantially affect the structure composed of other unsubstituted amino acids.
　基質結合部位に存在する第452番目及び第453番目のアミノ酸を、それぞれ上記のように置換することにより、酸性基質の認識を可能にする効果が得られる。また、第312番目～第315番目のアミノ酸、それぞれ上記のように置換することにより、GM2活性化因子との会合（結合）に必要なループ構造が導入できるという効果が得られる。GM2活性化因子は、酵素タンパク質と基質（GM2ガングリオシド）との邂逅に働くものであり、α-サブユニット由来の活性を発揮するために重要な因子である。 The effect of allowing recognition of an acidic substrate can be obtained by substituting the 452nd and 453rd amino acids present in the substrate binding site, respectively, as described above. Further, by substituting each of the 312st to 315th amino acids as described above, it is possible to obtain an effect that a loop structure necessary for association (binding) with the GM2 activator can be introduced. The GM2 activator acts on the trap between the enzyme protein and the substrate (GM2 ganglioside) and is an important factor for exerting the activity derived from the α-subunit.
　本発明のタンパク質（ホモ二量体を含む）はまた、以下の(a)又は(b)のタンパク質であることが好ましい。
　 (a) 下記(i)～(iv)のいずれかのアミノ酸配列を含むタンパク質。
　  (i) 配列番号４に示されるアミノ酸配列において第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換されたアミノ酸配列
　  (ii) 配列番号４に示されるアミノ酸配列において第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換されたアミノ酸配列
　  (iii) 配列番号４に示されるアミノ酸配列において第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列
　  (iv) 配列番号４に示されるアミノ酸配列において、第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換され、かつ第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換され、かつ第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列 The protein of the present invention (including homodimer) is also preferably the following protein (a) or (b).
(a) A protein comprising any one of the following amino acid sequences (i) to (iv):
(i) An amino acid in which the 312th to 315th amino acids in the amino acid sequence shown in SEQ ID NO: 4 are sequentially replaced with an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine. Sequence (ii) An amino acid sequence in which the 452nd amino acid is substituted with an amino acid other than aspartic acid in the amino acid sequence shown in SEQ ID NO: 4 (iii) In the amino acid sequence shown in SEQ ID NO: 4, the 453rd amino acid is other than leucine (Iv) in the amino acid sequence shown in SEQ ID NO: 4, the 312th to 315th amino acids are respectively an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and Substituted with an amino acid other than lysine and the 452nd position Amino acid is replaced by an amino acid other than aspartic acid, and the amino acid sequence # 453 amino acid has been substituted with an amino acid other than leucine
　 (b) 上記(i)～(iv)のいずれかのアミノ酸配列において前記置換部位のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質
　上記(a)のタンパク質としては、上記(i)及び(iv)の記載中の「アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸」が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであるタンパク質が好ましく挙げられる。同様に、上記(a)のタンパク質としては、上記(ii)及び(iv)の記載中の「アスパラギン酸以外のアミノ酸」がアスパラギンであるタンパク質や、上記(iii)及び(iv)の記載中の「ロイシン以外のアミノ酸」がアルギニンであるタンパク質も好ましく挙げられる。 (b) the amino acid sequence of any one of (i) to (iv) above, comprising an amino acid sequence in which one or several amino acids except the amino acid at the substitution site are deleted, substituted or added, and wild-type human β -Protein having activity derived from α-subunit of hexosaminidase As the protein of (a) above, the amino acids other than arginine, amino acids other than glutamine, other than asparagine in the description of (i) and (iv) above Preferred examples include proteins in which “amino acids other than lysine” are glycine, serine, glutamic acid, and proline, respectively. Similarly, as the protein of (a) above, a protein in which the `` amino acid other than aspartic acid '' in the description of (ii) and (iv) above is asparagine, or the protein in the description of (iii) and (iv) above. A protein in which the “amino acid other than leucine” is arginine is also preferred.
　上記(a)のタンパク質としては、上記(i)～(iv)のアミノ酸配列を含むタンパク質のうち、上記(iv)のアミノ酸配列を含むタンパク質がより好ましく、配列番号６に示されるアミノ酸配列を含むタンパク質がさらに好ましく、配列番号６に示されるアミノ酸配列からなるタンパク質が特に好ましい。 The protein (a) is more preferably a protein comprising the amino acid sequence (iv) among the proteins comprising the amino acid sequences (i) to (iv), and comprises the amino acid sequence represented by SEQ ID NO: 6. A protein is more preferred, and a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is particularly preferred.
　上記(b)のタンパク質は、上記(a)のタンパク質に含まれる上記(i)～(iv)のいずれかのアミノ酸配列において前記置換部位のアミノ酸を除く、１個又は数個（例えば１個～１０個程度、好ましくは１個～５個程度）のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含み、かつα-サブユニット由来の活性を有するタンパク質であればよく、限定はされない。当該欠失、置換若しくは付加は、β-サブユニットのシグナルペプチドを除く部分においてなされることが好ましい。当該シグナルペプチドは、配列番号４に示されるアミノ酸配列における第1番目～第54番目のアミノ酸からなる部分である。
　本発明においては、α-サブユニット由来の活性は、例えば、CHO細胞やヒト線維芽細胞等の哺乳類由来の細胞に目的タンパク質を発現させて採取し、4-MUGS分解活性を測定することにより確認できる。具体的には、当該タンパク質（酵素溶液）と、4-メチルウムベリフェリル-N-アセチル-β-D-グルコサミン-6-硫酸（4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide）（人工基質）とを混合して、pH4.5の条件下で反応させた場合に、当該酵素溶液の単位量が単位時間当たりに遊離させ得る4-メチルウムベリフェロン（4-methylumbelliferone）の量を検出することにより測定することができる。4-メチルウムベリフェロンの検出は、公知の各種検出方法を採用できるが、例えば、蛍光光度計等を用いて検出する方法が好ましい。また、目的タンパク質の発現は、公知の各種発現ベクター等に組込んで細胞に導入し発現させればよい。 The protein of (b) is one or several (for example, one to several) excluding the amino acid at the substitution site in any one of the amino acid sequences (i) to (iv) included in the protein (a). There is no limitation as long as the protein has an amino acid sequence in which about 10 (preferably about 1 to 5) amino acids are deleted, substituted or added and has an activity derived from the α-subunit. The deletion, substitution or addition is preferably performed in a portion excluding the signal peptide of the β-subunit. The signal peptide is a portion consisting of the first to 54th amino acids in the amino acid sequence shown in SEQ ID NO: 4.
In the present invention, the α-subunit-derived activity is confirmed by, for example, collecting and expressing the target protein in cells derived from mammals such as CHO cells and human fibroblasts, and measuring 4-MUGS degradation activity. it can. Specifically, the protein (enzyme solution) and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulfate (4-methylumbelliferyl-6-sulfo-N-acetyl-β-D- 4-methylumbelliferone that can be released per unit time when the enzyme solution is mixed with glucosaminide (artificial substrate) and reacted under pH 4.5 conditions. Can be measured by detecting the amount of. For the detection of 4-methylumbelliferone, various known detection methods can be adopted. For example, a detection method using a fluorometer or the like is preferable. The target protein may be expressed by incorporating it into various known expression vectors and introducing it into cells.
 
３．組換え遺伝子
　上述した本発明のタンパク質をコードする遺伝子としては、限定はされないが、以下の(a)又は(b)のDNAを含む遺伝子が好ましく挙げられる。なお、以下の(a)及び(b)のDNAは、いずれも本発明のタンパク質の構造遺伝子であることが好ましいが、これらDNAを含む遺伝子としては、これらDNAのみからなるものであってもよいし、これらDNAを一部に含み、その他に遺伝子発現に必要な公知の塩基配列（転写プロモーター、SD配列、Kozak配列、ターミネーター等）をも含むものであってもよく、限定はされない。
3. Recombinant gene The gene encoding the protein of the present invention described above is not limited, but a gene containing the following DNA (a) or (b) is preferably exemplified. The following DNAs (a) and (b) are preferably both structural genes of the protein of the present invention, but a gene containing these DNAs may be composed only of these DNAs. In addition, these DNAs may be included in part, and may also include known base sequences (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) necessary for gene expression, and are not limited.
　 (a) 下記(i)～(iv)のいずれかの塩基配列を含むDNA
　  (i) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (ii) 配列番号３に示される塩基配列において第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (iii) 配列番号３に示される塩基配列において第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
　  (iv) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換され、かつ第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換され、かつ第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列 (a) DNA comprising any one of the nucleotide sequences (i) to (iv) below
(i) In the base sequence shown in SEQ ID NO: 3, the 934th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases Were sequentially replaced with a base indicating an amino acid codon other than arginine, a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, respectively. Base sequence (ii) Base sequence in which the 1354th to 1356th bases in the base sequence shown in SEQ ID NO: 3 are replaced with bases indicating codons of amino acids other than aspartic acid (iii) Base shown in SEQ ID NO: 3 A base sequence in which the 1357th to 1359th bases in the sequence are replaced with bases indicating codons of amino acids other than leucine (iv) in the base sequence shown in SEQ ID NO: 3, The 34th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases, in order, have codons for amino acids other than arginine, respectively. Is replaced with a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, and the 1354th to 1356th bases are other than aspartic acid. A base sequence in which the bases 1357 to 1359 are substituted with bases indicating the codons of amino acids other than leucine.
　 (b) 上記(i)～(iv)のいずれかの塩基配列を含むDNAに対し相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、前記置換部位の塩基に対応する塩基が当該置換部位の塩基と同一であり、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質をコードするDNA
　本発明において「コドン」とは、転写後のRNA配列上の３塩基連鎖（トリプレット）に限らず、DNA配列上の３塩基連鎖をも意味する。よって、DNA配列上のコドンの表記は、ウラシル(U)の代わりにチミン(T)を用いて行う。
　配列番号３に示される塩基配列は、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニット（556アミノ酸）をコードする1671個の塩基からなる塩基配列である。 (b) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising any one of the base sequences (i) to (iv) above, wherein the base at the substitution site A DNA encoding a protein having the same base as that of the substitution site and an activity derived from the α-subunit of wild-type human β-hexosaminidase
In the present invention, the term “codon” means not only a three-base chain (triplet) on the RNA sequence after transcription but also a three-base chain on the DNA sequence. Therefore, the codon notation on the DNA sequence is performed using thymine (T) instead of uracil (U).
The base sequence shown in SEQ ID NO: 3 is a base sequence consisting of 1671 bases encoding the β-subunit (556 amino acids) of wild-type human β-hexosaminidase.
　また、上記(a)のDNAとしては、上記(i)及び(iv)の記載中の「アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基」が、それぞれ順に、グリシンのコドンを示す塩基、セリンのコドンを示す塩基、グルタミン酸のコドンを示す塩基及びプロリンのコドンを示す塩基である場合のDNAが好ましく挙げられる。同様に、上記(a)のDNAとしては、上記(ii)及び(iv)の記載中の「アスパラギン酸以外のアミノ酸のコドンを示す塩基」がアスパラギンのコドンを示す塩基である場合のDNAや、上記(iii)及び(iv)の記載中の「ロイシン以外のアミノ酸のコドンを示す塩基」がアルギニンのコドンを示す塩基である場合のDNAも好ましく挙げられる。 In addition, as the DNA of the above (a), in the description of the above (i) and (iv), “a base indicating a codon of an amino acid other than arginine, a base indicating a codon of an amino acid other than glutamine, a codon of an amino acid other than asparagine” And a base indicating a codon of amino acid other than lysine are a base indicating a codon of glycine, a base indicating a codon of serine, a base indicating a codon of glutamic acid, and a base indicating a codon of proline, respectively. DNA is preferred. Similarly, as the DNA of the above (a), in the description of the above (ii) and (iv) `` DNA that indicates the codon of the amino acid other than aspartic acid '' is a base that indicates the codon of asparagine, Preferable examples also include DNAs in which the “bases indicating the codons of amino acids other than leucine” in the descriptions of (iii) and (iv) above are bases indicating the codons of arginine.
　ここで、上記の各アミノ酸のコドンを示す塩基（左端の塩基を５’側の塩基とする）については、グリシンのコドンを示す塩基は「ggt」、「ggc」、「gga」又は「ggg」（好ましくは「ggg」）であり、セリンのコドンを示す塩基は「tct」、「tcc」、「tca」、「tcg」、「agt」又は「agc」（好ましくは「tct」）であり、グルタミン酸のコドンを示す塩基は「gag」又は「gaa」（好ましくは「gag」）であり、プロリンのコドンを示す塩基は「cct」、「ccc」、「cca」又は「ccg」（好ましくは「ccc」）であり、アスパラギン（但し、上記アスパラギン酸以外のアミノ酸としてのもの）のコドンを示す塩基は「aat」又は「aac」（好ましくは「aac」）であり、アルギニンのコドンを示す塩基は「cgt」、「cgc」、「cga」、「cgg」、「aga」又は「agg」（好ましくは「cgt」）である。
　上記(a)のDNAとしては、上記(i)～(iv)の塩基配列を含むDNAのうち、上記(iv)の塩基配列を含むDNAがより好ましく、配列番号５に示される塩基配列を含むDNAがさらに好ましく、配列番号５に示される塩基配列からなるDNAが特に好ましい。 Here, for the bases indicating the codons of the above amino acids (the leftmost base is the 5 ′ base), the bases indicating the glycine codons are “ggt”, “ggc”, “gga”, or “ggg”. (Preferably “ggg”) and the base indicating the codon of serine is “tct”, “tcc”, “tca”, “tcg”, “agt” or “agc” (preferably “tct”), The base indicating the codon for glutamic acid is “gag” or “gaa” (preferably “gag”), and the base indicating the codon for proline is “cct”, “ccc”, “cca” or “ccg” (preferably “ ccc "), the base indicating the codon of asparagine (but as an amino acid other than the above aspartic acid) is" aat "or" aac "(preferably" aac "), and the base indicating the codon of arginine is “Cgt”, “cgc”, “cga”, “cgg”, “aga” or “agg” (preferably “cgt” ).
The DNA of the above (a) is more preferably a DNA containing the base sequence of the above (iv) among the DNAs containing the base sequences of the above (i) to (iv), including the base sequence shown in SEQ ID NO: 5. DNA is more preferred, and DNA consisting of the base sequence shown in SEQ ID NO: 5 is particularly preferred.
　以上のような変異置換型のDNAは、例えば、Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) 等に記載の部位特異的変位誘発法に準じて調製することができる。具体的には、Kunkel法や Gapped duplex法等の公知手法により、部位特異的突然変異誘発法を利用した変異導入用キットを用いて調製することができ、当該キットとしては、例えば、QuickChangeTMSite-Directed Mutagenesis Kit（ストラタジーン社製）、GeneTailorTMSite-Directed Mutagenesis System（インビトロジェン社製）、TaKaRa Site-Directed Mutagenesis System（Mutan-K、Mutan-Super Express Km等：タカラバイオ社製）等が好ましく挙げられる。 Such mutation-substituted DNA is described in, for example, Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. It can be prepared according to the site-specific displacement induction method. Specifically, it can be prepared using a mutagenesis kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, and examples of the kit include QuickChange TM Site. -Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) are preferred Can be mentioned.
　また、所望のアミノ酸のコドンを示す塩基となるようにミスセンス変異が導入されるように設計したPCRプライマーを用い、β-サブユニットをコードする塩基配列を含むDNA等をテンプレートとして、適当な条件下でPCRを行うことにより調製することもできる。PCRに用いるDNAポリメラーゼは、限定はされないが、正確性の高いDNAポリメラーゼであることが好ましく、例えば、Pwo DNA（ポリメラーゼロシュ・ダイアグノスティックス）、Pfu DNAポリメラーゼ（プロメガ）、プラチナPfx DNAポリメラーゼ（インビトロジェン）、KOD DNAポリメラーゼ（東洋紡）、KOD-plus-ポリメラーゼ（東洋紡）等が好ましい。PCRの反応条件は、用いるDNAポリメラーゼの最適温度、合成するDNAの長さや種類等により適宜設定すればよいが、例えば、サイクル条件であれば「90～98℃で5～30秒（熱変性・解離）→50～65℃で5～30秒（アニーリング）→65～80℃で30～1200秒（合成・伸長）」を１サイクルとして合計20～200サイクル行う条件が好ましい。 In addition, using PCR primers designed to introduce a missense mutation so that a base representing the codon of the desired amino acid is introduced, using a DNA containing a base sequence encoding the β-subunit as a template under appropriate conditions It can also be prepared by PCR. Although the DNA polymerase used for PCR is not limited, it is preferably a highly accurate DNA polymerase. For example, Pwo DNA (Polymerase Roche Diagnostics), Pfu DNA polymerase (Promega), Platinum Pfx DNA polymerase ( Invitrogen), KOD DNA polymerase (Toyobo), KOD-plus-polymerase (Toyobo) and the like are preferable. The PCR reaction conditions may be appropriately set depending on the optimum temperature of the DNA polymerase to be used, the length and type of DNA to be synthesized, etc. For example, in the case of cycle conditions, “90 to 98 ° C. for 5 to 30 seconds (thermal denaturation / Dissociation) → 50 to 65 ° C. for 5 to 30 seconds (annealing) → 65 to 80 ° C. for 30 to 1200 seconds (synthesis / elongation) ”is preferably performed under a total of 20 to 200 cycles.
　上記(b)のDNAは、上記(i)～(iv)のいずれかの塩基配列を含むDNA（すなわち上記(a)のDNA）若しくはそれと相補的な塩基配列からなるDNA、又はこれらを断片化したものをプローブとして用い、コロニーハイブリダイゼーション、プラークハイブリダイゼーション、及びサザンブロット等の公知のハイブリダイゼーション法を実施し、cDNAライブラリーやゲノムライブラリーから得ることができる。ライブラリーは、公知の方法で作製されたものを利用してもよいし、市販のcDNAライブラリーやゲノムライブラリーを利用してもよく、限定はされない。
　ハイブリダイゼーション法の詳細な手順については、Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)等を適宜参照することができる。
　ハイブリダイゼーション法を実施における「ストリンジェントな条件」とは、ハイブリダイゼーション後の洗浄時の条件であって、バッファーの塩濃度が15～330mM、温度が25～65℃、好ましくは塩濃度が15～150mM、温度が45～55℃の条件を意味する。具体的には、例えば80mMで50℃等の条件を挙げることができる。さらに、このような塩濃度や温度等の条件に加えて、プローブ濃度、プローブの長さ、反応時間等の諸条件も考慮し、上記(b)のDNAを得るための条件を適宜設定することができる。 The DNA of (b) is a DNA comprising any of the nucleotide sequences (i) to (iv) (that is, the DNA of (a) above), a DNA comprising a complementary nucleotide sequence, or a fragment thereof. The obtained product can be used as a probe and subjected to known hybridization methods such as colony hybridization, plaque hybridization, and Southern blot, and can be obtained from a cDNA library or a genomic library. A library prepared by a known method may be used, or a commercially available cDNA library or genomic library may be used, and is not limited.
For detailed procedures of the hybridization method, Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) can be referred to as appropriate.
“Stringent conditions” in carrying out the hybridization method are conditions at the time of washing after hybridization, wherein the buffer salt concentration is 15 to 330 mM, the temperature is 25 to 65 ° C., preferably the salt concentration is 15 to It means the condition of 150mM and temperature of 45 ~ 55 ℃. Specifically, for example, conditions such as 50 mM at 80 mM can be mentioned. Furthermore, in addition to such conditions as salt concentration and temperature, various conditions such as probe concentration, probe length, reaction time, etc. are also considered, and conditions for obtaining the DNA of (b) above should be set as appropriate. Can do.
　ハイブリダイズするDNAとしては、上記(a)のDNAの塩基配列に対して少なくとも40％以上の相同性を有する塩基配列であることが好ましく、より好ましくは60％、さらに好ましくは90％以上、特に好ましくは95％以上、最も好ましくは99％以上である。
　また、上記(b)のDNAは、前記置換部位の塩基に対応する塩基が当該置換部位の塩基と同一である。ここで、「前記置換部位の塩基に対応する塩基」の「対応する塩基」とは、上記(b)のDNAが上記(a)のDNAに対する相補鎖とハイブリダイズした場合に、このハイブリッドにおいて、前記置換部位の塩基に対する相補塩基（トリプレット）と、位置的に対向する関係にある塩基（トリプレット）を意味する。例えば、上記(b)のDNAの塩基配列が、上記(a)のDNAと比較して欠失及び付加の変異が無い場合（つまり両DNAの長さ(塩基数)が同じ）であれば、上記(b)のDNAの塩基配列における、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基、及び第943番目～第945番目の塩基、並びに、第1354番目～第1356番目の塩基、及び第1357番目～第1359番目の塩基が、上記「対応する塩基」となる。 The hybridizing DNA is preferably a base sequence having at least 40% homology with the DNA base sequence of (a) above, more preferably 60%, even more preferably 90% or more, particularly Preferably it is 95% or more, Most preferably, it is 99% or more.
In the DNA of (b) above, the base corresponding to the base at the substitution site is the same as the base at the substitution site. Here, the “corresponding base” of the “base corresponding to the base of the substitution site” means that when the DNA of (b) is hybridized with a complementary strand to the DNA of (a), It means a base (triplet) which is in a position opposite to the base complementary to the base at the substitution site (triplet). For example, if the base sequence of the DNA of (b) is free of deletion and addition mutations compared to the DNA of (a) (that is, the length (number of bases) of both DNAs is the same), In the base sequence of the DNA of (b) above, the 934th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases The bases, the 1354th to 1356th bases, and the 1357th to 1359th bases are the “corresponding bases”.
　さらに、上記(b)のDNAは、β-サブユニットのシグナルペプチドをコードする塩基配列領域が、上記(a)のDNAと同一であるものが好ましい。当該シグナルペプチドをコードする塩基配列領域は、配列番号３に示される塩基配列における第1番目～第162番目の塩基からなる領域である。
　上記(b)のDNAとしては、例えば、上記(a)のDNAと比較して、塩基配列については完全に同一ではないが、翻訳された後のアミノ酸配列については完全に同一となるような塩基配列からなるDNA（すなわち上記(a)のDNAにサイレント変異が施されたDNA）が、特に好ましい。
　本発明のタンパク質をコードする遺伝子としては、翻訳後の個々のアミノ酸に対応するコドンは、特に限定はされないので、転写後、ヒト等の哺乳類において一般的に用いられているコドン（好ましくは使用頻度の高いコドン）を示すDNAを含むものであってもよいし、また、大腸菌や酵母等の微生物や、植物等において一般的に用いられているコドン（好ましくは使用頻度の高いコドン）を示すDNAを含むものであってもよい。 Further, the DNA of (b) is preferably one in which the base sequence region encoding the signal peptide of the β-subunit is the same as the DNA of (a). The base sequence region encoding the signal peptide is a region composed of the first to 162nd bases in the base sequence shown in SEQ ID NO: 3.
As the DNA of (b) above, for example, the base sequence is not completely the same as the DNA of (a) above but the amino acid sequence after translation is completely the same. A DNA consisting of a sequence (that is, a DNA obtained by subjecting the DNA of (a) to a silent mutation) is particularly preferred.
As the gene encoding the protein of the present invention, codons corresponding to individual amino acids after translation are not particularly limited. Therefore, codons generally used in mammals such as humans after transcription (preferably frequency of use) DNA that shows a codon that is commonly used in microorganisms such as E. coli and yeast, plants, etc. (preferably a codon that is frequently used). May be included.
 
４．組換えベクター及び形質転換体
　本発明のタンパク質を発現させるためには、まず、上述した本発明の遺伝子を発現ベクターに組込んで組換えベクターを構築することが必要である。この際、発現ベクターに組込む遺伝子には、必要に応じて、予め、上流に転写プロモーター、SD配列（宿主が原核細胞の場合）及びKozak配列（宿主が真核細胞の場合）を連結しておいてもよいし、下流にターミネーターを連結しておいてもよく、その他、エンハンサー、スプライシングシグナル、ポリＡ付加シグナル、選択マーカー等を連結しておくこともできる。なお、上記転写プロモーター等の遺伝子発現に必要な各要素は、初めから当該遺伝子に含まれていてもよいし、もともと発現ベクターに含まれている場合はそれを利用してもよく、各要素の使用態様は特に限定されない。
　発現ベクターに当該遺伝子を組込む方法としては、例えば、制限酵素を用いる方法や、トポイソメラーゼを用いる方法など、公知の遺伝子組換え技術を利用した各種方法が採用できる。また、発現ベクターとしては、例えば、プラスミドDNA、バクテリオファージDNA、レトロトランスポゾンDNA、レトロウイルスベクター、人工染色体DNAなど、本発明のタンパク質をコードする遺伝子を保持し得るものであれば、限定はされず、使用する宿主細胞に適したベクターを適宜選択して使用することができる。
4). Recombinant Vector and Transformant In order to express the protein of the present invention, it is first necessary to construct a recombinant vector by incorporating the above-described gene of the present invention into an expression vector. At this time, a transcription promoter, SD sequence (when the host is a prokaryotic cell) and Kozak sequence (when the host is a eukaryotic cell) are ligated upstream in advance as necessary to the gene to be incorporated into the expression vector. Alternatively, a terminator may be linked downstream, and an enhancer, splicing signal, poly A addition signal, selection marker, etc. may be linked. Each element necessary for gene expression such as the above transcription promoter may be included in the gene from the beginning, or may be used when originally included in the expression vector. A use aspect is not specifically limited.
As a method for incorporating the gene into the expression vector, various methods using known gene recombination techniques such as a method using a restriction enzyme and a method using topoisomerase can be employed. The expression vector is not limited as long as it can hold the gene encoding the protein of the present invention, such as plasmid DNA, bacteriophage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, and the like. A vector suitable for the host cell to be used can be appropriately selected and used.
　次いで、構築した上記組換えベクターを宿主に導入して形質転換体を得、これを培養することにより、本発明のタンパク質を発現させることができる。なお、本発明で言う「形質転換体」とは宿主に外来遺伝子が導入されたものを意味し、例えば、宿主にプラスミドDNA等を導入すること（形質転換）で外来遺伝子が導入されたもの、並びに、宿主に各種ウイルス及びファージを感染させること（形質導入）で外来遺伝子が導入されたものが含まれる。
　宿主としては、上記組換えベクターが導入された後、本発明のタンパク質を発現し得るものであれば、限定はされず、適宜選択することができるが、例えば、ヒトやマウス等の各種動物細胞、各種植物細胞、細菌、酵母、植物細胞等の公知の宿主が挙げられる。
　動物細胞を宿主とする場合は、例えば、ヒト繊維芽細胞、CHO細胞、ベイビーハムスター腎臓由来の培養細胞（BHK細胞）サル細胞COS-7、Vero、マウスL細胞、ラットGH3、ヒトFL細胞等が用いられる。また、Sf9細胞、Sf21細胞等の昆虫細胞を用いることもできる。 Subsequently, the constructed recombinant vector is introduced into a host to obtain a transformant, which is cultured, whereby the protein of the present invention can be expressed. The “transformant” as used in the present invention means a gene into which a foreign gene has been introduced into the host, for example, a gene into which a foreign gene has been introduced by introducing plasmid DNA or the like into the host (transformation), Also included are those in which a foreign gene has been introduced by infecting a host with various viruses and phages (transduction).
The host is not limited as long as it can express the protein of the present invention after the introduction of the above recombinant vector, and can be selected as appropriate. For example, various animal cells such as humans and mice can be selected. And known hosts such as various plant cells, bacteria, yeast, and plant cells.
When animal cells are used as hosts, for example, human fibroblasts, CHO cells, baby hamster kidney-derived cultured cells (BHK cells) monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells, etc. Used. Insect cells such as Sf9 cells and Sf21 cells can also be used.
　細菌を宿主とする場合、例えば、大腸菌、枯草菌等が用いられる。
　酵母を宿主とする場合は、例えば、サッカロミセス・セレビシエ（Saccharomyces cerevisiae）、シゾサッカロミセス・ポンベ（Schizosaccharomyces pombe）等が用いられる。
　植物細胞を宿主とする場合は、例えば、タバコBY-2細胞等が用いられる。
　形質転換体を得る方法は、限定はされず、宿主と発現ベクターとの種類の組み合わせを考慮し、適宜選択することができるが、例えば、電気穿孔法、リポフェクション法、ヒートショック法、PEG法、リン酸カルシウム法、DEAEデキストラン法、並びに、DNAウイルスやRNAウイルス等の各種ウイルスを感染させる方法などが好ましく挙げられる。
　得られる形質転換体においては、組換えベクターに含まれる遺伝子のコドン型は、実際に用いた宿主のコドン型と一致していてもよいし、異なっていてもよく、限定はされない。 When bacteria are used as hosts, for example, Escherichia coli, Bacillus subtilis and the like are used.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used.
When plant cells are used as hosts, for example, tobacco BY-2 cells are used.
The method for obtaining the transformant is not limited, and can be appropriately selected in consideration of the combination of the host and the expression vector. For example, electroporation, lipofection, heat shock, PEG, Preferred examples include calcium phosphate method, DEAE dextran method, and methods of infecting various viruses such as DNA virus and RNA virus.
In the resulting transformant, the codon type of the gene contained in the recombinant vector may be the same as or different from the codon type of the host actually used, and is not limited.
 
５．タンパク質の製法
　本発明のタンパク質は、
野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位（特に基質結合部位）の構造を、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニットの基質が結合できるように変化させ、かつGM2ガングリオシド活性化因子と結合し得る構造（ループ構造；α-サブユニットに存在する構造）を導入することにより製造することができる。このような構造変化は、例えば、遺伝子組換え技術等により、前述したようにβ-サブユニットを構成するアミノ酸配列中の特定のアミノ酸残基を他のアミノ酸残基に置換することにより行うことができる。
　本発明のタンパク質の製造は、具体的には、前述した形質転換体を培養する工程と、得られる培養物からα-サブユニット由来の活性を有するタンパク質を採取する工程とを含む方法により実施することができる。ここで、「培養物」とは、培養上清、培養細胞、培養菌体、又は細胞若しくは菌体の破砕物のいずれをも意味するものである。上記形質転換体の培養は、宿主の培養に用いられる通常の方法に従って行うことができる。目的のタンパク質は、上記培養物中に蓄積される。
5). Protein production method The protein of the present invention comprises:
The structure of the active site (especially the substrate binding site) of the wild-type human β-hexosaminidase β-subunit is changed so that the substrate of the wild-type human β-hexosaminidase α-subunit can bind. And a structure capable of binding to the GM2 ganglioside activator (loop structure; structure existing in the α-subunit). Such a structural change can be performed, for example, by substituting a specific amino acid residue in the amino acid sequence constituting the β-subunit with another amino acid residue, as described above, by a genetic recombination technique or the like. it can.
Specifically, the production of the protein of the present invention is carried out by a method including the step of culturing the above-described transformant and the step of collecting a protein having activity derived from α-subunit from the obtained culture. be able to. Here, “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The transformant can be cultured according to a usual method used for host culture. The protein of interest is accumulated in the culture.
　上記培養に用いる培地としては、宿主が資化し得る炭素源、窒素源、無機塩類などを含有し、形質転換体の培養を効率的に行うことができる培地であれば、公知の各種天然培地及び合成培地のいずれを用いてもよい。
　培養中は、形質転換体に含まれる組換えベクターの脱落及び目的タンパク質をコードする遺伝子の脱落を防ぐために、選択圧をかけた状態で培養してもよい。すなわち、選択マーカーが薬剤耐性遺伝子である場合には、相当する薬剤を培地に添加することができ、選択マーカーが栄養要求性相補遺伝子である場合には、相当する栄養因子を培地から除くことができる。例えば、G418耐性遺伝子を含むベクターで形質導入したヒト線維芽細胞を培養する場合、培養中、必要に応じてG418（G418硫酸塩）を添加してもよい。
　プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した形質転換体等を培養する場合は、必要に応じて、好適なインデューサー（例えば、IPTG等）を培地に添加してもよい。 As the medium used for the culture, any known natural medium and any known medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host, and can efficiently culture the transformant. Any synthetic medium may be used.
During the culture, the cells may be cultured under selective pressure in order to prevent the recombinant vector contained in the transformant from dropping and the gene encoding the target protein from dropping off. That is, when the selectable marker is a drug resistance gene, the corresponding drug can be added to the medium, and when the selectable marker is an auxotrophic complementary gene, the corresponding nutrient factor can be removed from the medium. it can. For example, when culturing human fibroblasts transduced with a vector containing a G418 resistance gene, G418 (G418 sulfate) may be added as needed during the culture.
When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, a suitable inducer (eg, IPTG) may be added to the medium as necessary.
　形質転換体の培養条件は、目的タンパク質の生産性及び宿主の生育が妨げられない条件であれば特に限定はされず、通常、10℃～40℃、好ましくは20℃～37℃で5～100時間行う。pHの調整は、無機又は有機酸、アルカリ溶液等を用いて行うことができる。培養方法としては、固体培養、静置培養、振盪培養、通気攪拌培養などが挙げられる。
　培養後、目的タンパク質が菌体内又は細胞内に生産される場合には、菌体又は細胞を破砕することにより目的タンパク質を採取することができる。菌体又は細胞の破砕方法としては、フレンチプレス又はホモジナイザーによる高圧処理、超音波処理、ガラスビーズ等による磨砕処理、リゾチーム、セルラーゼ又はペクチナーゼ等を用いる酵素処理、凍結融解処理、低張液処理、ファージによる溶菌誘導処理等を利用することができる。破砕後、必要に応じて菌体又は細胞の破砕残渣（細胞抽出液不溶性画分を含む）を除くことができる。残渣を除去する方法としては、例えば、遠心分離やろ過などが挙げられ、必要に応じて、凝集剤やろ過助剤等を使用して残渣除去効率を上げることもできる。残渣を除去した後に得られた上清は、細胞抽出液可溶性画分であり、粗精製したタンパク質溶液とすることができる。 The culture conditions of the transformant are not particularly limited as long as the productivity of the target protein and the growth of the host are not hindered, and are usually 10 ° C to 40 ° C, preferably 20 ° C to 37 ° C, and 5 to 100. Do time. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.
When the target protein is produced in the microbial cells or cells after culturing, the target protein can be collected by disrupting the microbial cells or cells. As a method for disrupting cells or cells, high-pressure treatment using a French press or homogenizer, ultrasonic treatment, grinding treatment using glass beads, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, hypotonic solution treatment, It is possible to use a lysis inducing treatment with a phage or the like. After crushing, the cells or cell crushing residues (including the cell extract insoluble fraction) can be removed as necessary. Examples of the method for removing the residue include centrifugation and filtration. If necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid. The supernatant obtained after removing the residue is a cell extract soluble fraction and can be a crudely purified protein solution.
　また、目的のタンパク質が菌体内又は細胞内に生産される場合は、菌体や細胞そのものを遠心分離、膜分離等で回収して、未破砕のまま使用することも可能である。
　一方、目的のタンパク質が菌体外又は細胞外に生産される場合には、培養液をそのまま使用するか、遠心分離やろ過等により菌体又は細胞を除去する。その後、必要に応じて硫安沈澱による抽出等により、培養物中から目的タンパク質を採取し、さらに必要に応じて透析、各種クロマトグラフィー（ゲルろ過、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等）を用いて単離精製することもできる。
　形質転換体等を培養して得られたタンパク質の生産収率は、例えば、培養液あたり、菌体湿重量又は乾燥重量あたり、粗酵素液タンパク質あたりなどの単位で、SDS-PAGE（ポリアクリルアミドゲル電気泳動）等により確認することができる。
　また、目的タンパク質の製造は、上述したような形質転換体を用いたタンパク質合成系のほか、生細胞を全く使用しない無細胞タンパク質合成系を用いて行うこともできる。
　無細胞タンパク質合成系とは、細胞抽出液を用いて試験管等の人工容器内で目的タンパク質を合成する系である。また、使用し得る無細胞タンパク質合成系としては、DNAを鋳型としてRNAを合成する無細胞転写系も含まれる。 In addition, when the target protein is produced in the microbial cells or cells, the microbial cells and the cells themselves can be recovered by centrifugation, membrane separation, etc., and used without being crushed.
On the other hand, when the target protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or filtration. Then, if necessary, the target protein is collected from the culture by extraction with ammonium sulfate precipitation, and further, if necessary, using dialysis and various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.) It can also be isolated and purified.
The production yield of the protein obtained by culturing the transformant is, for example, SDS-PAGE (polyacrylamide gel) in units such as per culture solution, per microbial wet weight or dry weight, or per crude enzyme solution protein. For example, electrophoresis).
In addition to the protein synthesis system using the transformant as described above, the target protein can also be produced using a cell-free protein synthesis system that does not use any living cells.
The cell-free protein synthesis system is a system that synthesizes a target protein in an artificial container such as a test tube using a cell extract. Cell-free protein synthesis systems that can be used also include cell-free transcription systems that synthesize RNA using DNA as a template.
　この場合、使用する細胞抽出液の由来は、前述の宿主細胞であることが好ましい。細胞抽出液としては、例えば真核細胞由来又は原核細胞由来の抽出液、より具体的には、CHO細胞、ウサギ網状赤血球、マウスL-細胞、HeLa細胞、小麦胚芽、出芽酵母、大腸菌などの抽出液を使用することができる。なお、これらの細胞抽出液は、濃縮又は希釈して用いてもよいし、そのままでもよく、限定はされない。
　細胞抽出液は、例えば限外濾過、透析、ポリエチレングリコール(PEG)沈殿等によって得ることができる。
　このような無細胞タンパク質合成は、市販のキットを用いて行うこともできる。例えば、試薬キットPROTEIOSTM（東洋紡）、TNTTM System（プロメガ）、合成装置のPG-MateTM（東洋紡）、RTS（ロシュ・ダイアグノスティクス）等が挙げられる。
　無細胞タンパク質合成によって産生された目的のタンパク質は、前述したようにクロマトグラフィー等の手段を適宜選択して、精製することができる。 In this case, the cell extract to be used is preferably derived from the aforementioned host cell. Examples of cell extracts include extracts derived from eukaryotic cells or prokaryotic cells. More specifically, CHO cells, rabbit reticulocytes, mouse L-cells, HeLa cells, wheat germ, budding yeast, Escherichia coli, and the like are extracted. Liquid can be used. These cell extracts may be used after being concentrated or diluted, or may be used as they are, and are not limited.
The cell extract can be obtained, for example, by ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like.
Such cell-free protein synthesis can also be performed using a commercially available kit. For example, reagent kits PROTEIOS ™ (Toyobo), TNT ™ System (Promega), synthesizer PG-Mate ™ (Toyobo), RTS (Roche Diagnostics) and the like.
The target protein produced by cell-free protein synthesis can be purified by appropriately selecting means such as chromatography as described above.
 
６．医薬組成物
　(i) 補充用酵素薬等としての医薬組成物
　本発明のタンパク質は、前述したように、テイ－サックス病及びザンドホッフ病の治療に関して種々の優れた効果を発揮し得るものであり、テイ－サックス病治療剤及びザンドホッフ病治療剤の有効成分として用いることができる。すなわち、本発明は、前述した本発明のタンパク質を含有するテイ－サックス病治療用医薬組成物（テイ－サックス病治療薬）及びザンドホッフ病治療用医薬組成物（ザンドホッフ病治療薬）を提供するものである。これらの医薬組成物としては、具体的には、酵素補充療法に用い得る補充用酵素薬が好ましい。なお、これらの医薬組成物に用いる本発明のタンパク質としては、ホモ二量体であるもの（すなわち改変Hex B）が特に好ましい。
　当該医薬組成物において有効成分となる、本発明のタンパク質は、必要に応じて各種塩や水和物等の状態で用いられてもよいし、また、治療剤としての保存安定性（特に活性維持）を考慮し適当な化学的修飾がなされた状態で用いられてもよく、限定はされない。
6). Pharmaceutical composition (i) Pharmaceutical composition as a replenishment enzyme, etc. As described above, the protein of the present invention can exert various excellent effects on the treatment of Tay-Sachs disease and Sandhoff disease. It can be used as an active ingredient of a therapeutic agent for Tay-Sachs disease and a therapeutic agent for Sandhoff disease. That is, the present invention provides a pharmaceutical composition for the treatment of Tay-Sachs disease (Tea-Sachs disease therapeutic agent) and a pharmaceutical composition for the treatment of Sandhoff disease (A therapeutic agent for Sandhoff disease) containing the protein of the present invention described above. It is. Specifically, these pharmaceutical compositions are preferably enzyme enzymes for replenishment that can be used for enzyme replacement therapy. The protein of the present invention used in these pharmaceutical compositions is particularly preferably a homodimer (ie, modified Hex B).
The protein of the present invention, which is an active ingredient in the pharmaceutical composition, may be used in the state of various salts, hydrates, etc., if necessary, and also has storage stability as a therapeutic agent (particularly activity maintenance). ) May be used in a state in which appropriate chemical modification is made, and is not limited.
　当該医薬組成物は、本発明のタンパク質等以外にも他の成分を含むことができる。他の成分としては、当該医薬組成物の用法（使用形態）に応じて必要とされる製薬上の各種成分（薬学的に許容し得る各種担体等）が挙げられる。他の成分は、本発明のタンパク質等により発揮される効果が損なわれない範囲で適宜含有することができる。
　当該医薬組成物が補充用酵素薬に用いられる場合は、本発明のタンパク質の配合割合や、他の成分の種類及び配合割合は、公知の補充用酵素薬の調製法に準じて適宜設定することができる。
　当該医薬組成物の投与については、その用法は限定はされないが、補充用酵素薬である場合は、通常、点滴静注などの非経口用法が採用される。非経口用法等の各種用法に用い得る製剤は、薬剤製造上一般に用いられる賦形剤、充填剤、増量剤、結合剤、湿潤剤、崩壊剤、潤滑剤、界面活性剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、矯味矯臭剤、無痛化剤、安定化剤、等張化剤等を適宜選択して使用し、常法により調製することができる。 The pharmaceutical composition can contain other components besides the protein of the present invention. Examples of the other components include various pharmaceutical components (various pharmaceutically acceptable carriers and the like) required depending on the usage (form of use) of the pharmaceutical composition. Other components can be appropriately contained as long as the effects exhibited by the protein of the present invention are not impaired.
When the pharmaceutical composition is used as a replenishment enzyme, the proportion of the protein of the present invention and the types and proportions of other components should be set appropriately according to known methods for preparing replenishment enzymes. Can do.
The administration of the pharmaceutical composition is not limited, but in the case of a supplementary enzyme, parenteral methods such as intravenous infusion are usually employed. The preparations that can be used for various methods such as parenteral methods include excipients, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, and buffering agents that are commonly used in pharmaceutical production. Preservatives, solubilizers, preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and used, and can be prepared by conventional methods.
　当該医薬組成物の形態は、限定はされないが、補充用酵素薬である場合は、通常、静脈内注射剤（点滴を含む）が採用され、例えば、単位投与量アンプル又は多投与量容器の状態等で提供され得る。
　当該医薬組成物の投与量は、一般には、製剤中の有効成分の配合割合を考慮した上で、投与対象（患者）の年齢、体重、病気の種類、病状のほか、投与経路、投与回数、投与期間等を勘案し、適宜、広範囲に設定することができる。特に、本発明の治療剤が補充用酵素薬である場合、その投与回数は、２～４週間に１回程度が好ましく、またその投与量（／１回）は、例えば、有効成分である本発明のタンパク質等（組換え酵素）を、患者の体重に対して0.1～10mg/kg程度投与できる量であることが好ましく、より好ましくは0.1～5mg/kg程度、さらに好ましくは0.2～1mg/kg程度である。 The form of the pharmaceutical composition is not limited, but in the case of a replenishing enzyme drug, an intravenous injection (including infusion) is usually adopted, for example, a unit dose ampoule or a multi-dose container state Etc. may be provided.
The dosage of the pharmaceutical composition is generally determined in consideration of the compounding ratio of the active ingredient in the preparation, and the administration subject (patient) age, weight, type of disease, medical condition, administration route, number of administrations, A wide range can be set as appropriate in consideration of the administration period and the like. In particular, when the therapeutic agent of the present invention is a supplementary enzyme drug, the number of administrations is preferably about once every 2 to 4 weeks, and the dosage (/ once) is, for example, the active ingredient. The amount of the protein or the like (recombinant enzyme) of the invention is preferably such that it can be administered at about 0.1 to 10 mg / kg, more preferably about 0.1 to 5 mg / kg, still more preferably 0.2 to 1 mg / kg relative to the body weight of the patient. Degree.
　本発明においては、有効成分となる本発明のタンパク質（組換え酵素）は、血中安定性に優れ、障害臓器の細胞への取り込み効率も高いため、少量の使用であっても従来基準と同様又はそれ以上の優れた酵素補充効果を得ることができ、またアレルギー性副作用等の有害作用も極めて少ないので、患者への体力的、精神的及び経済的な負担を大いに低減することができる。 In the present invention, the protein of the present invention (recombinant enzyme) as an active ingredient is excellent in blood stability and has a high efficiency of uptake into cells of damaged organs. In addition, an excellent enzyme supplementation effect can be obtained, and since there are very few adverse effects such as allergic side effects, the physical, mental and economic burden on the patient can be greatly reduced.
　(ii) 遺伝子治療剤としての医薬組成物
　本発明の遺伝子は、前述したように、テイ－サックス病及びザンドホッフ病の治療に関して種々の優れた効果を発揮し得る本発明のタンパク質をコードするものであり、テイ－サックス病治療用医薬組成物（テイ－サックス病治療薬（具体的には遺伝子治療薬））及びザンドホッフ病治療用医薬組成物（ザンドホッフ病治療薬（具体的には遺伝子治療薬））の有効成分として用いることができる。
　当該医薬組成物（遺伝子治療剤）を用いる場合は、注射により直接投与する方法のほか、核酸が組込まれたベクターを投与する方法が挙げられる。上記ベクターとしては、アデノウイルスベクター、アデノ関連ウイルスベクター、ヘルペスウイルスベクター、ワクシニアウイルスベクター、レトロウイルスベクター及びレンチウイルスベクター等が挙げられる。これらのウイルスベクターを用いることにより効率よく投与することができる。なお、市販の遺伝子導入キット（例えば、製品名：アデノエクスプレス、クローンテック社製）を用いることもできる。 (ii) Pharmaceutical Composition as a Gene Therapeutic Agent As described above, the gene of the present invention encodes the protein of the present invention that can exert various excellent effects on the treatment of Tay-Sachs disease and Sandhoff disease. Yes, Tey-Sachs disease treatment pharmaceutical composition (Tey-Sachs disease treatment (specifically gene therapy agent)) and Zandhoff disease treatment pharmaceutical composition (Zandhoff disease treatment agent (specifically gene therapy agent)) ) As an active ingredient.
When using the said pharmaceutical composition (gene therapy agent), the method of administering the vector in which the nucleic acid was integrated other than the method of administering directly by injection is mentioned. Examples of the vector include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, and lentivirus vectors. It can administer efficiently by using these viral vectors. A commercially available gene transfer kit (for example, product name: Adeno Express, manufactured by Clontech) can also be used.
　また、当該医薬組成物（遺伝子治療薬）を用いる場合、当該組成物をリポソーム等のリン脂質小胞体に導入し、この小胞体を投与することも可能である。本発明の遺伝子を保持させた小胞体をリポフェクション法により所定の細胞に導入する。そして、得られる細胞を例えば静脈内又は動脈内等に投与する。ファブリー病の障害臓器に局所投与することもできる。例えば、成人に当該医薬組成物を投与する場合は、患者の体重に対し、一日あたり0.1μg/kg～1000mg/kg程度であることが好ましく、より好ましくは1μg/kg～100mg/kg程度である。 In addition, when the pharmaceutical composition (gene therapy drug) is used, it is also possible to introduce the composition into a phospholipid vesicle such as a liposome and administer the vesicle. The endoplasmic reticulum holding the gene of the present invention is introduced into a predetermined cell by the lipofection method. Then, the obtained cells are administered, for example, intravenously or intraarterially. It can also be administered locally to an organ affected by Fabry disease. For example, when the pharmaceutical composition is administered to an adult, it is preferably about 0.1 μg / kg to 1000 mg / kg per day, more preferably about 1 μg / kg to 100 mg / kg relative to the weight of the patient. is there.
 
７．治療方法
　本発明は、上述した医薬組成物をテイ－サックス病患者やザンドホッフ病患者に投与することを特徴とする、テイ－サックス病の治療方法及びザンドホッフ病の治療方法を含むものである。また本発明は、テイ－サックス病又はザンドホッフ病を治療するための上記医薬組成物又は本発明のタンパク質及び／若しくは遺伝子の使用、並びに、テイ－サックス病又はザンドホッフ病の治療のための薬剤を製造するための上記医薬組成物又は本発明のタンパク質及び／若しくは遺伝子の使用も含む。
　本発明の治療方法に使用する医薬組成物は、本発明のタンパク質を含む医薬組成物（前記「６.(i)」；補充用酵素薬）、又は本発明の遺伝子を含む医薬組成物（前記「６.(ii)；遺伝子治療薬」）、あるいはこれら両医薬組成物の併用であってもよく、限定はされず、患者の病状や副作用の有無、あるいは投与効果などを考慮し、適宜選択することができる。
　特に、上記併用の場合は、それぞれの医薬組成物の投与量の割合、投与回数及び投与期間などを、個々の患者に合わせて適宜設定することができる。なお、各医薬組成物等の好ましい投与方法及び投与量等については、前述の通りである。
7). Therapeutic Method The present invention includes a method for treating Tay-Sachs disease and a method for treating Sandhoff disease, characterized by administering the above-described pharmaceutical composition to a Tay-Sachs disease patient or a Sandhoff disease patient. The present invention also provides the use of the above pharmaceutical composition or the protein and / or gene of the present invention for the treatment of Tay-Sachs disease or Sandhoff disease, and the manufacture of a medicament for the treatment of Tay-Sachs disease or Sandhoff disease. This includes the use of the above pharmaceutical composition or the protein and / or gene of the present invention.
The pharmaceutical composition used in the therapeutic method of the present invention is a pharmaceutical composition containing the protein of the present invention (the above “6. (i)”; a supplementary enzyme drug), or a pharmaceutical composition containing the gene of the present invention (the above-mentioned “6. (ii); gene therapy drug”), or a combination of both of these pharmaceutical compositions, which is not limited, and is appropriately selected in consideration of the patient's medical condition, the presence or absence of side effects, the administration effect, etc. can do.
In particular, in the case of the above combination, the dose ratio, the number of administrations, the administration period, and the like of each pharmaceutical composition can be appropriately set according to individual patients. In addition, about the preferable administration method and dosage etc. of each pharmaceutical composition etc., it is as above-mentioned.
　以下に、実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
＜ヒトβ-ヘキソサミニダーゼAのα-サブユニットの機能領域＞
　ヒトβ-ヘキソサミニダーゼBの基質特異性をβ-ヘキソサミニダーゼAのそれに変換し、GM2ガングリオシドを加水分解する機能を持つ新規酵素を設計するため、タンパク質立体構造モデルを用いた検討を行った。図１に示されるように、ヒトβ-ヘキソサミニダーゼAは、α-サブユニットとβ-サブユニットから構成される。GM2ガングリオシドを基質として認識する部位及びGM2ガングリオシドと酵素とを会合させるGM2活性化因子との結合部位は、α-サブユニットにあると考えられた。 <Functional region of α-subunit of human β-hexosaminidase A>
In order to convert the substrate specificity of human β-hexosaminidase B to that of β-hexosaminidase A and design a new enzyme with the function of hydrolyzing GM2 ganglioside, studies using a protein three-dimensional structure model were conducted. went. As shown in FIG. 1, human β-hexosaminidase A is composed of an α-subunit and a β-subunit. It was considered that the site that recognizes GM2 ganglioside as a substrate and the binding site of GM2 activator that associates GM2 ganglioside with an enzyme are in the α-subunit.
　そこで、Protein Data Bank (PDB)に登録されているヒトβ-ヘキソサミニダーゼAの立体構造データ(PDB ID: 2GJX)と生化学的解析情報に基づいて、活性ポケットを構成する15アミノ酸残基（R178, H204, D207, H262, D322, E323, W373, W392, Y421, N423, R424, W460, G461, E462, Y423）、GM2活性化因子との結合に関与する可変的ループを構成する4アミノ酸残基（G280, S281, E282, P283）、及びβ-サブユニットとの2量体形成に関与する39アミノ酸残基（R178, H179, Y180, P209, Y227, N228, T231, H232, N423, R424, I425, S426, Y427, G428, P429, E462, Y463, V464, D465, T467, N468, P471, R472, R504, L508, Q513, A514, Q515, P516, L517, N518, V519, G520, F521, C522, E523, E525, F526, E527）を特定した。その結果を図２に示した。なお、ここで用いたアミノ酸残基の表記は、α-サブユニットのアミノ酸配列（配列番号２）中のアミノ酸残基の種類と位置を示しており、例えば「R178」は、配列番号２で示されるアミノ酸配列の第178番目のアルギニン残基を示すものである。 Therefore, based on the three-dimensional structure data of human β-hexosaminidase A (PDB ID: 2GJX) registered in Protein Data Bank (PDB) and biochemical analysis information, 15 amino acid residues constituting the active pocket (R178, H204, D207, H262, D322, E323, W373, W392, Y421, N423, R424, W460, G461, E462, Y423), 4 amino acids that constitute a variable loop involved in binding to GM2 activator Residues (G280, S281, E282, P283) and 39 amino acid residues involved in dimer formation with β-subunit (R178, H179, Y180, P209, Y227, N228, T231, H232, N423, R424 , I425, S426, Y427, G428, P429, E462, Y463, V464, D465, T467, N468, P471, R472, R504, L513, A514, Q515, P516, L517, N518, V519, G520, F521 , E523, E525, F526, E527). The results are shown in FIG. The amino acid residue notation used here indicates the type and position of the amino acid residue in the amino acid sequence of the α-subunit (SEQ ID NO: 2). For example, “R178” is indicated by SEQ ID NO: 2. 178th arginine residue of the amino acid sequence shown.
＜GM2ガングリオシドの分解に必要なβ-サブユニットのアミノ酸置換＞
　β-サブユニットにおいて、酸性基質の認識を可能にするアミノ酸置換（D452N，L453R）、及びGM2活性化因子との会合に必要なループ構造の導入に伴うアミノ酸置換（RQNK (312-315) GSEP）を行った。
　実施例１で得られたα-サブユニット上の機能的領域を構成するアミノ酸残基に関して、それぞれに対応するβ-サブユニット上のアミノ酸残基を特定した。α-サブユニットとβ-サブユニットのアミノ酸配列及び立体構造の異同について比較した。基質GM2ガングリオシドを認識させるために、β-サブユニット中の第452番目のアミノ酸残基（D）をα-サブユニット中の当該アミノ酸残基に対応するアミノ酸残基（N）に置換し、かつβ-サブユニット中の第453番目のアミノ酸残基（L）をα-サブユニット中の当該アミノ酸残基に対応するアミノ酸残基（R）に置換した。また、GM2活性化因子と結合させるために、β-サブユニット中の第312番目～第315番目のアミノ酸配列（RQNK）をα-サブユニット中の当該アミノ酸配列に対応するアミノ酸配列（GSEP）に置換した。その結果を図３に示した。なお、このようにアミノ酸置換した改変β-サブユニットのアミノ酸配列は、配列番号６に示されるアミノ酸配列である。 <Amino acid substitution of β-subunit required for degradation of GM2 ganglioside>
In the β-subunit, amino acid substitution that enables recognition of acidic substrates (D452N, L453R), and amino acid substitution that accompanies the introduction of the loop structure required for association with GM2 activator (RQNK (312-315) GSEP) Went.
Regarding the amino acid residues constituting the functional region on the α-subunit obtained in Example 1, the corresponding amino acid residues on the β-subunit were identified. Differences in amino acid sequence and three-dimensional structure of α-subunit and β-subunit were compared. In order to recognize the substrate GM2 ganglioside, the 452nd amino acid residue (D) in the β-subunit is substituted with an amino acid residue (N) corresponding to the amino acid residue in the α-subunit, and The 453rd amino acid residue (L) in the β-subunit was substituted with an amino acid residue (R) corresponding to the amino acid residue in the α-subunit. In order to bind to the GM2 activator, the amino acid sequence (RQNK) from 312 to 315 in the β-subunit is changed to the amino acid sequence (GSEP) corresponding to the amino acid sequence in the α-subunit. Replaced. The results are shown in FIG. The amino acid sequence of the modified β-subunit that has been amino acid substituted in this way is the amino acid sequence shown in SEQ ID NO: 6.
＜細胞株の樹立＞
　図４に示される手順及び遺伝子組換え技術の常法に従い、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのcDNAを、哺乳類発現ベクターpCXN2のマルチクローニングサイトに挿入し、組換え発現ベクターpCXN2／HEXBを構築した。
　併せて、pCXN2／HEXBベクター中のβ-サブユニットのcDNAに塩基置換を行って改変β-サブユニットのcDNAとした、組換え発現ベクターpCXN2／mut-HEXBを構築した。塩基置換は、β-サブユニットのアミノ酸配列中の第452番目のアミノ酸D（アスパラギン酸）がN（アスパラギン）に、第453番目のアミノ酸L（ロイシン）がR（アルギニン）に、第312番目～第315番目のアミノ酸配列RQNK（アルギニン-グルタミン-アスパラギン-リジン）がGSEP（グリシン-セリン-グルタミン酸-プロリン）に置換されるように各アミノ酸残基に対応するコドンの塩基を置換した。具体的には、上記アミノ酸Dに対応するコドン「gat」をNに対応する「aac」に置換し、上記アミノ酸Lに対応するコドン「ttg」をRに対応する「cgt」に置換し、上記アミノ酸配列RQNKに対応する4つのコドン「aga caa aac aag」をGSEPに対応する「ggg tct gag ccc」に置換した。なお、このように塩基置換した改変β-サブユニットをコードするcDNAの塩基配列は、配列番号５に示される塩基配列である。 <Establishment of cell lines>
In accordance with the procedure shown in FIG. 4 and the conventional method of gene recombination technology, the cDNA of the wild-type human β-hexosaminidase β-subunit is inserted into the multicloning site of the mammalian expression vector pCXN 2 for recombinant expression. vectors were constructed pCXN 2 / HEXB.
In addition, to the cDNA of pCXN 2 / HEXB vector of β- subunit cDNA modified β- subunits carried out base substitutions in the constructed a recombinant expression vector pCXN 2 / mut-HEXB. In the amino acid sequence of the β-subunit, the 452nd amino acid D (aspartic acid) is replaced with N (asparagine), the 453rd amino acid L (leucine) is replaced with R (arginine), the 312th to The base of the codon corresponding to each amino acid residue was substituted so that the 315th amino acid sequence RQNK (arginine-glutamine-asparagine-lysine) was replaced with GSEP (glycine-serine-glutamic acid-proline). Specifically, the codon “gat” corresponding to the amino acid D is replaced with “aac” corresponding to N, the codon “ttg” corresponding to the amino acid L is replaced with “cgt” corresponding to R, and Four codons “aga caa aac aag” corresponding to the amino acid sequence RQNK were replaced with “ggg tct gag ccc” corresponding to GSEP. The base sequence of the cDNA encoding the modified β-subunit thus base-substituted is the base sequence shown in SEQ ID NO: 5.
　次いで、pCXN2／HEXB及びpCXN2／mut-HEXBを、それぞれCHO細胞に導入し、ネオマイシン誘導体（G418硫酸塩）の存在下で各々の遺伝子を恒常発現する薬剤耐性細胞集団を選別した。さらに、限界希釈法を用いて各々の遺伝子を高発現するCHOクローン細胞株を樹立した。 Subsequently, pCXN 2 / HEXB and pCXN 2 / mut-HEXB were each introduced into CHO cells, and drug-resistant cell populations that constantly express each gene in the presence of a neomycin derivative (G418 sulfate) were selected. Furthermore, a CHO clone cell line that highly expresses each gene was established using a limiting dilution method.
＜CHO細胞抽出液及び培養上清におけるHex活性＞
　実施例３で得られた薬剤耐性細胞集団（60mm dish）を10%牛胎児血清存在下で3日間培養後、細胞抽出液を調製した。また、同細胞集団を無血清培地中で３日間培養後、その培養上清を回収した。図５に、細胞抽出液及び培養上清中の、4-MUG分解酵素活性（図５中、黒塗り表示）及び4-MUGS分解酵素活性（図５中、黒斜線表示）の測定結果を示した。野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットcDNAを発現するCHO細胞集団では高い4-MUG分解活性の増大が観察されたが、4-MUGS分解活性の増大は認められなかった。一方、pCXN2／mut-HEXBを導入した細胞集団では、4-MUG分解活性の増大のみならず、有意な4-MUGS分解活性の増大が認められた。
　なお、「4-MUG」は、4-メチルウムベリフェリル-N-アセチル-β-D-グルコサミン（4-methylumbelliferyl-N-acetyl-β-D-glucosaminide）を意味し、Hex B活性測定の際に人工基質として用いられるものである。一方、「4-MUGS」は、4-メチルウムベリフェリル-N-アセチル-β-D-グルコサミン-6-硫酸（4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide）を意味し、Hex A活性測定の際に人工基質として用いられるものである。 <Hex activity in CHO cell extract and culture supernatant>
The drug-resistant cell population (60 mm dish) obtained in Example 3 was cultured for 3 days in the presence of 10% fetal bovine serum, and a cell extract was prepared. The cell population was cultured in a serum-free medium for 3 days, and the culture supernatant was collected. Fig. 5 shows the measurement results of 4-MUG degrading enzyme activity (shown in black in Fig. 5) and 4-MUGS degrading enzyme activity (shown in black diagonal lines in Fig. 5) in the cell extract and culture supernatant. It was. A high increase in 4-MUG degradation activity was observed in the CHO cell population expressing wild-type human β-hexosaminidase β-subunit cDNA, but no increase in 4-MUGS degradation activity was observed. On the other hand, in the cell population was introduced pCXN 2 / mut-HEXB, 4 -MUG not increased degradation activity alone, significant increase in 4-Mugs degradation activity was observed.
“4-MUG” means 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide, which is used when measuring Hex B activity. It is used as an artificial substrate. On the other hand, “4-MUGS” means 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide However, it is used as an artificial substrate when measuring Hex A activity.
＜細胞抽出液及び培養上清における改変Hex Bの発現＞
　実施例４で得られた、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットcDNA及びその改変型cDNAを発現する各々のCHO細胞集団の抽出液中に存在するHexアイソザイムを、非変性条件下ポリアクリルアミドゲル電気泳動及び抗ヒトHex Aポリクローナル抗体を用いた、イムノブロッティングにより解析した。その結果を図６に示した。野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットcDNAを発現するCHO細胞の抽出液及び培養上清では、Hex Bに対応するブロードなバンドのみが観察され、Hex Aは検出されなかった。また改変型cDNAを発現する細胞の場合も、Hex Bを移動度の異なるバンドは観察されたが、Hex Aとは異なる移動度を示す改変Hex Bが検出された。 <Expression of modified Hex B in cell extract and culture supernatant>
The Hex isozyme present in the extract of each CHO cell population expressing the β-subunit cDNA of wild-type human β-hexosaminidase and its modified cDNA obtained in Example 4 was subjected to non-denaturing conditions. Analysis was performed by immunoblotting using lower polyacrylamide gel electrophoresis and anti-human Hex A polyclonal antibody. The results are shown in FIG. Only a broad band corresponding to Hex B was observed in the extract and culture supernatant of CHO cells expressing the β-subunit cDNA of wild-type human β-hexosaminidase, and Hex A was not detected. In the case of cells expressing the modified cDNA, bands having different mobility from Hex B were observed, but modified Hex B having a mobility different from Hex A was detected.
＜培養線維芽細胞への補充実験に用いた各Hexアイソザイム画分の粗精製＞
　10mM NaPiB (pH 6.0)で平衡化した陰イオン交換体（DEAE）カラムを用いて、野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニット及び改変型cDNAを発現するCHO細胞抽出液から各Hexアイソザイムを分離した。NaClを用いたグラジエント溶出により、CHO由来のHex Aは200mM NaCl画分に、ヒトHex B及び改変Hex Bはflow- through画分にそれぞれ回収された。これらの粗精製画分を、10mM NaPiB (pH 6.0)でバッファー交換し、患者由来繊維芽細胞への補充実験に用いた。 <Rough purification of each Hex isozyme fraction used for supplementation to cultured fibroblasts>
Each Hex from CHO cell extracts expressing wild-type human β-hexosaminidase β-subunit and modified cDNA using an anion exchanger (DEAE) column equilibrated with 10 mM NaPiB (pH 6.0) Isoenzymes were isolated. By gradient elution using NaCl, CHO-derived Hex A was recovered in the 200 mM NaCl fraction, and human Hex B and modified Hex B were recovered in the flow-through fraction. These crudely purified fractions were buffer exchanged with 10 mM NaPiB (pH 6.0), and used for a supplementation experiment on patient-derived fibroblasts.
＜改変Hex B投与によるGM2ガングリオシドの分解効果＞
　ザンドホッフ病患者由来の培養線維芽細胞の培養液中に、野生型ヒトHex A（4-MUGS分解活性：500 nmol/h）、野生型ヒトHex B（4-MUG分解活性：900 nmol/h）及び改変Hex B（4-MUGS分解活性：2,000 nmol/h）を加えて3日間培養後に細胞を固定し、抗GM2ガングリオシド抗体を用いて免疫蛍光染色を行った。その結果を図７に示した。野生型Hex Aを投与した場合、GM2ガングリオシドの顕著な減少が観察されたのに対し、野生型Hex Bを投与した場合はGM2の分解はほとんど認められなかった。一方、改変型Hex Bを投与した場合は、野生型Hex Aの4倍量の4-MUGS分解活性を示し、顕著なGM2の分解が認められた。 <Decomposition effect of GM2 ganglioside by modified Hex B administration>
Wild-type human Hex A (4-MUGS degradation activity: 500 nmol / h), wild-type human Hex B (4-MUG degradation activity: 900 nmol / h) in the culture fluid of cultured fibroblasts from patients with Zandhoff disease Then, modified Hex B (4-MUGS degradation activity: 2,000 nmol / h) was added, the cells were fixed after culturing for 3 days, and immunofluorescent staining was performed using an anti-GM2 ganglioside antibody. The results are shown in FIG. When wild-type Hex A was administered, a significant decrease in GM2 ganglioside was observed, whereas when wild-type Hex B was administered, almost no degradation of GM2 was observed. On the other hand, when modified Hex B was administered, 4-MUGS degradation activity was 4 times that of wild-type Hex A, and significant degradation of GM2 was observed.
＜改変Hex Bの性質＞
　β-ヘキソサミニダーゼのアイソザイムであるβ-ヘキソサミニダーゼA(Hex A)と、β-ヘキソサミニダーゼB(Hex B)とを比較した場合、基質であるGM2ガングリオシドを加水分解する機能はHex Aのみが有し、HexBは持たず、また、熱に対する安定性は、両者を比較するとHex Bの方が著しく高いことが知られている。Hex A及びHex Bは、共に糖タンパク質で、コアとなるタンパク質部分に糖鎖が付いているが、その個数は、Hex Aでは6本、Hex Bでは8本であることが知られている。酵素の細胞内取り込みは、糖鎖末端に存在するマンノース6-リン酸の数によって規定されるため、糖鎖数が多い方が、取り込みに有利と考えられた。改変Hex Bは、基質認識部位とGM2活性化因子との結合部位をHex A様に変化させているため、GM2ガングリオシド分解能を有していた。また、分子のほとんどがHex B様であり、糖鎖結合部位は変化させていないため、安定性はHex Bと同様に、30%野生型マウス血漿中、37℃での安定性もHex Aに比べて高かった。さらに糖鎖数も多いため、細胞内取り込みに優れていると考えられた。 <Properties of modified Hex B>
The ability to hydrolyze the substrate GM2 ganglioside when comparing β-hexosaminidase A (Hex A), which is an isozyme of β-hexosaminidase, with β-hexosaminidase B (Hex B) Has only Hex A, no HexB, and it is known that Hex B has significantly higher heat stability than both. Hex A and Hex B are both glycoproteins, and the number of sugar chains is known to be 6 in Hex A and 8 in Hex B. Since the cellular uptake of the enzyme is defined by the number of mannose 6-phosphate present at the sugar chain end, it was considered that the larger number of sugar chains is more advantageous for the uptake. The modified Hex B had GM2 ganglioside resolution because the binding site between the substrate recognition site and the GM2 activator was changed to Hex A-like. In addition, since most of the molecules are Hex B-like and the sugar chain binding site is not changed, the stability is similar to Hex B, and the stability at 37 ° C in 30% wild-type mouse plasma is also Hex A. It was higher than that. Furthermore, since there were many sugar chains, it was thought that it was excellent in cellular uptake.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜結論＞
　以上の実施例より、改変Hex Bは、テイ－サックス病やザンドホッフ病の患者の体内に蓄積するGM2ガングリオシドを分解する機能を持ち、その投与により発生頻度の高いテイ－サックス病患者にアレルギー反応を起こす危険性が少なく、野生型Hex Aよりも安定性が高く、細胞内取り込みに優れるものであったため、これらの疾患の治療薬として有望であることが分かった。 <Conclusion>
From the above examples, the modified Hex B has the function of degrading GM2 ganglioside accumulated in the body of patients with Tay-Sachs disease and Sandhoff disease, and allergic reaction is caused to Tay-Sachs disease patients who frequently occur by its administration. It was found to be promising as a therapeutic agent for these diseases because it has a lower risk of occurrence, is more stable than wild-type Hex A, and excels in cellular uptake.
配列番号５：組換えDNA
配列番号６：組換えタンパク質 Sequence number 5: Recombinant DNA
SEQ ID NO: 6: recombinant protein

Claims (32)

  1. 　野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を変化させて、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を獲得したタンパク質。 Protein that has acquired the activity derived from the α-subunit of wild-type human β-hexosaminidase by changing the structure of the active site of the β-subunit of wild-type human β-hexosaminidase.
  2. 　野生型ヒトβ-ヘキソサミニダーゼのα-サブユニットの基質特異性を有する、請求項１記載のタンパク質。 The protein according to claim 1, which has the substrate specificity of the α-subunit of wild-type human β-hexosaminidase.
  3. 　野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列において第312番目～第315番目、第452番目及び第453番目のアミノ酸のうち少なくとも１つのアミノ酸が他のアミノ酸に置換されたアミノ酸配列、又は当該置換されたアミノ酸配列のうち第312番目～第315番目、第452番目及び第453番目のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有する、タンパク質。 An amino acid in which at least one of amino acids 312 to 315, 452 and 453 in the amino acid sequence of the β-subunit of wild-type human β-hexosaminidase is substituted with another amino acid A sequence, or an amino acid sequence in which one or several amino acids except amino acids 312 to 315, 452 and 453 of the substituted amino acid sequence are deleted, substituted or added, A protein having activity derived from the α-subunit of wild-type human β-hexosaminidase.
  4. 　第312番目～第315番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換された、請求項３記載のタンパク質。 4. The protein according to claim 3, wherein the 312th to 315th amino acids are substituted with glycine, serine, glutamic acid and proline, respectively.
  5. 　第452番目のアミノ酸がアスパラギンに置換された、請求項３又は４記載のタンパク質。 The protein according to claim 3 or 4, wherein the amino acid at position 452 is substituted with asparagine.
  6. 　第453番目のアミノ酸がアルギニンに置換された、請求項３～５のいずれか１項に記載のタンパク質。 The protein according to any one of claims 3 to 5, wherein the 453rd amino acid is substituted with arginine.
  7. 　第312番目～第315番目のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換され、かつ第452番目のアミノ酸がアスパラギンに置換され、かつ第453番目のアミノ酸がアルギニンに置換された、請求項３記載のタンパク質。 The 312th to 315th amino acids were sequentially replaced with glycine, serine, glutamic acid and proline, the 452nd amino acid was replaced with asparagine, and the 453rd amino acid was replaced with arginine. The protein according to claim 3.
  8. 　以下の(a)又は(b)のタンパク質。
    　 (a) 下記(i)～(iv)のいずれかのアミノ酸配列を含むタンパク質。
    　  (i) 配列番号４に示されるアミノ酸配列において第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換されたアミノ酸配列
    　  (ii) 配列番号４に示されるアミノ酸配列において第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換されたアミノ酸配列
    　  (iii) 配列番号４に示されるアミノ酸配列において第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列
    　  (iv) 配列番号４に示されるアミノ酸配列において、第312番目～第315番目のアミノ酸が、それぞれ順に、アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸に置換され、かつ第452番目のアミノ酸がアスパラギン酸以外のアミノ酸に置換され、かつ第453番目のアミノ酸がロイシン以外のアミノ酸に置換されたアミノ酸配列
    　 (b) 上記(i)～(iv)のいずれかのアミノ酸配列において前記置換部位のアミノ酸を除く１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質     The following protein (a) or (b).
    (a) A protein comprising any one of the following amino acid sequences (i) to (iv):
    (i) An amino acid in which the 312th to 315th amino acids in the amino acid sequence shown in SEQ ID NO: 4 are sequentially replaced with an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and an amino acid other than lysine. Sequence (ii) An amino acid sequence in which the 452nd amino acid is substituted with an amino acid other than aspartic acid in the amino acid sequence shown in SEQ ID NO: 4 (iii) In the amino acid sequence shown in SEQ ID NO: 4, the 453rd amino acid is other than leucine (Iv) in the amino acid sequence shown in SEQ ID NO: 4, the 312th to 315th amino acids are respectively an amino acid other than arginine, an amino acid other than glutamine, an amino acid other than asparagine, and Substituted with an amino acid other than lysine and the 452nd position An amino acid sequence in which a mino acid is substituted with an amino acid other than aspartic acid and the 453rd amino acid is substituted with an amino acid other than leucine; (b) the substitution site in the amino acid sequence of any one of (i) to (iv) above; A protein comprising an amino acid sequence in which one or several amino acids except amino acids are deleted, substituted or added, and having an activity derived from the α-subunit of wild-type human β-hexosaminidase
  9. 　前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンである、請求項８記載のタンパク質。 The protein according to claim 8, wherein the amino acid other than arginine, the amino acid other than glutamine, the amino acid other than asparagine, and the amino acid other than lysine are glycine, serine, glutamic acid, and proline, respectively.
  10. 　前記アスパラギン酸以外のアミノ酸がアスパラギンである、請求項８又は９記載のタンパク質。 The protein according to claim 8 or 9, wherein the amino acid other than aspartic acid is asparagine.
  11. 　前記ロイシン以外のアミノ酸がアルギニンである、請求項８～１０のいずれか１項に記載のタンパク質。 The protein according to any one of claims 8 to 10, wherein the amino acid other than leucine is arginine.
  12. 　前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであり、かつ前記アスパラギン酸以外のアミノ酸がアスパラギンであり、かつ前記ロイシン以外のアミノ酸がアルギニンである、請求項８記載のタンパク質。 The amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine and amino acids other than lysine are glycine, serine, glutamic acid and proline, respectively, and the amino acid other than aspartic acid is asparagine, and the leucine. The protein according to claim 8, wherein the amino acid other than is arginine.
  13. 　請求項１～１２のいずれか１項に記載のタンパク質のホモ二量体からなる、タンパク質。 A protein comprising the homodimer of the protein according to any one of claims 1 to 12.
  14. 　請求項１～１２のいずれか１項に記載のタンパク質をコードする遺伝子。 A gene encoding the protein according to any one of claims 1 to 12.
  15. 　以下の(a)又は(b)のDNAを含む遺伝子。
    　 (a) 下記(i)～(iv)のいずれかの塩基配列を含むDNA
    　  (i) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
    　  (ii) 配列番号３に示される塩基配列において第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換された塩基配列
    　  (iii) 配列番号３に示される塩基配列において第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
    　  (iv) 配列番号３に示される塩基配列において、第934番目～第936番目の塩基、第937番目～第939番目の塩基、第940番目～第942番目の塩基及び第943番目～第945番目の塩基が、それぞれ順に、アルギニン以外のアミノ酸のコドンを示す塩基、グルタミン以外のアミノ酸のコドンを示す塩基、アスパラギン以外のアミノ酸のコドンを示す塩基及びリシン以外のアミノ酸のコドンを示す塩基に置換され、かつ第1354番目～第1356番目の塩基がアスパラギン酸以外のアミノ酸のコドンを示す塩基に置換され、かつ第1357番目～第1359番目の塩基がロイシン以外のアミノ酸のコドンを示す塩基に置換された塩基配列
    　 (b) 上記(i)～(iv)のいずれかの塩基配列を含むDNAに対し相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、前記置換部位の塩基に対応する塩基が当該置換部位の塩基と同一であり、かつ野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質をコードするDNA     A gene comprising the following DNA (a) or (b):
    (a) DNA comprising any one of the nucleotide sequences (i) to (iv) below
    (i) In the base sequence shown in SEQ ID NO: 3, the 934th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases Were sequentially replaced with a base indicating an amino acid codon other than arginine, a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, respectively. Base sequence (ii) Base sequence in which the 1354th to 1356th bases in the base sequence shown in SEQ ID NO: 3 are replaced with bases indicating codons of amino acids other than aspartic acid (iii) Base shown in SEQ ID NO: 3 A base sequence in which the 1357th to 1359th bases in the sequence are replaced with bases indicating codons of amino acids other than leucine (iv) in the base sequence shown in SEQ ID NO: 3, The 34th to 936th bases, the 937th to 939th bases, the 940th to 942nd bases, and the 943rd to 945th bases, in order, have codons for amino acids other than arginine, respectively. Is replaced with a base indicating an amino acid codon other than glutamine, a base indicating an amino acid codon other than asparagine, and a base indicating an amino acid codon other than lysine, and the 1354th to 1356th bases are other than aspartic acid. (B) any one of (i) to (iv) above, wherein the nucleotides 1357 to 1359 are replaced with bases indicating amino acid codons other than leucine. A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising any of the base sequences, wherein the base corresponding to the base of the substitution site is DNA encoding a protein that is identical to the base and has an activity derived from the α-subunit of wild-type human β-hexosaminidase
  16. 　前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンである、請求項１５記載の遺伝子。 The gene according to claim 15, wherein the amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine, and amino acids other than lysine are glycine, serine, glutamic acid, and proline, respectively.
  17. 　前記アスパラギン酸以外のアミノ酸がアスパラギンである、請求項１５又は１６記載の遺伝子。 The gene according to claim 15 or 16, wherein the amino acid other than aspartic acid is asparagine.
  18. 　前記ロイシン以外のアミノ酸がアルギニンである、請求項１５～１７のいずれか１項に記載の遺伝子。 The gene according to any one of claims 15 to 17, wherein the amino acid other than leucine is arginine.
  19. 　前記アルギニン以外のアミノ酸、グルタミン以外のアミノ酸、アスパラギン以外のアミノ酸及びリシン以外のアミノ酸が、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンであり、かつ前記アスパラギン酸以外のアミノ酸がアスパラギンであり、かつ前記ロイシン以外のアミノ酸がアルギニンである、請求項１５記載の遺伝子。 The amino acids other than arginine, amino acids other than glutamine, amino acids other than asparagine and amino acids other than lysine are glycine, serine, glutamic acid and proline, respectively, and the amino acid other than aspartic acid is asparagine, and the leucine. The gene according to claim 15, wherein the amino acid other than is arginine.
  20. 　請求項１４～１９のいずれか１項に記載の遺伝子を含む組換えベクター。 A recombinant vector comprising the gene according to any one of claims 14 to 19.
  21. 　請求項２０記載の組換えベクターを含む形質転換体。 A transformant comprising the recombinant vector according to claim 20.
  22. 　野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を変化させることにより、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を付与することを特徴とする、酵素の基質特異性変換方法。 It is characterized by conferring an activity derived from the α-subunit of wild-type human β-hexosaminidase by changing the structure of the active site of the β-subunit of wild-type human β-hexosaminidase Method for converting substrate specificity of enzyme.
  23. 　野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列における第312番目～第315番目のアミノ酸を、それぞれ順に、グリシン、セリン、グルタミン酸及びプロリンに置換し、かつ第452番目のアミノ酸をアスパラギンに置換し、かつ第453番目のアミノ酸をアルギニンに置換することにより、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を付与することを特徴とする、請求項２２記載の方法。 In the amino acid sequence of the β-subunit of wild-type human β-hexosaminidase, the 312st to 315th amino acids are respectively replaced with glycine, serine, glutamic acid and proline, and the 452nd amino acid is replaced. 23. The activity derived from the α-subunit of wild-type human β-hexosaminidase is imparted by substituting asparagine and substituting the 453rd amino acid with arginine. Method.
  24. 　前記野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットのアミノ酸配列が配列番号４に示されるアミノ酸配列である、請求項２３記載の方法。 The method according to claim 23, wherein the amino acid sequence of the β-subunit of the wild type human β-hexosaminidase is the amino acid sequence shown in SEQ ID NO: 4.
  25. 　野生型ヒトβ-ヘキソサミニダーゼのβ-サブユニットの活性部位の構造を、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニットの基質が結合できるように変化させることを特徴とする、野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質の製造方法。 Characterized in that the structure of the active site of the β-subunit of wild-type human β-hexosaminidase is changed so that the substrate of the α-subunit of wild-type human β-hexosaminidase can bind. A method for producing a protein having an activity derived from the α-subunit of wild-type human β-hexosaminidase.
  26. 　請求項２１記載の形質転換体を培養する工程と、得られる培養物から野生型ヒトβ-ヘキソサミニダーゼのα-サブユニット由来の活性を有するタンパク質を採取する工程とを含む、当該タンパク質の製造方法。 A step of culturing the transformant according to claim 21, and a step of collecting a protein having an activity derived from the α-subunit of wild-type human β-hexosaminidase from the obtained culture. Production method.
  27. 　請求項１～１３のいずれか１項に記載のタンパク質を含むことを特徴とする、テイ－サックス病治療用医薬組成物。 A pharmaceutical composition for treating Tay-Sachs disease comprising the protein according to any one of claims 1 to 13.
  28. 　請求項１４～１９のいずれか１項に記載の遺伝子を含むことを特徴とする、テイ－サックス病治療用医薬組成物。 A pharmaceutical composition for treating Tay-Sachs disease comprising the gene according to any one of claims 14 to 19.
  29. 　請求項２７記載の医薬組成物及び／又は請求項２８記載の医薬組成物をテイ－サックス病患者に投与することを特徴とする、テイ－サックス病の治療方法。 A method for treating Tay-Sachs disease, comprising administering the pharmaceutical composition according to Claim 27 and / or the pharmaceutical composition according to Claim 28 to a Tay-Sachs disease patient.
  30. 　請求項１～１３のいずれか１項に記載のタンパク質を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。 A pharmaceutical composition for treating Sandhoff's disease comprising the protein according to any one of claims 1 to 13.
  31. 　請求項１４～１９のいずれか１項に記載の遺伝子を含むことを特徴とする、ザンドホッフ病治療用医薬組成物。 A pharmaceutical composition for treating Sandhoff's disease comprising the gene according to any one of claims 14 to 19.
  32. 　請求項３０記載の医薬組成物及び／又は請求項３１記載の医薬組成物をザンドホッフ病患者に投与することを特徴とする、ザンドホッフ病の治療方法。
          A method for treating Sandhoff disease, comprising administering the pharmaceutical composition according to Claim 30 and / or the pharmaceutical composition according to Claim 31 to a patient with Sandhoff disease.
PCT/JP2010/050397 2009-01-16 2010-01-15 NOVEL HIGH-FUNCTION ENZYME OBTAINED BY ALTERING SUBSTRATE SPECIFICITY OF HUMAN β-HEXOSAMINIDASE Β WO2010082622A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2010546654A JP5678664B2 (en) 2009-01-16 2010-01-15 A novel high-performance enzyme that converts the substrate specificity of human β-hexosaminidase B

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009008039 2009-01-16
JP2009-008039 2009-01-16

Publications (1)

Publication Number Publication Date
WO2010082622A1 true WO2010082622A1 (en) 2010-07-22

Family

ID=42339876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/050397 WO2010082622A1 (en) 2009-01-16 2010-01-15 NOVEL HIGH-FUNCTION ENZYME OBTAINED BY ALTERING SUBSTRATE SPECIFICITY OF HUMAN β-HEXOSAMINIDASE Β

Country Status (2)

Country Link
JP (1) JP5678664B2 (en)
WO (1) WO2010082622A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014061735A1 (en) 2012-10-19 2014-04-24 国立大学法人徳島大学 NOVEL HIGH-FUNCTIONING ENZYME HAVING ALTERED HUMAN β-HEXOSAMINIDASE B SUBSTRATE SPECIFICITY, AND HAVING PROTEASE RESISTANCE APPLIED THERETO
WO2015150922A3 (en) * 2014-03-17 2016-01-28 The Hospital For Sick Children β-HEXOSAMINIDASE PROTEIN VARIANTS AND ASSOCIATED METHODS FOR TREATING GM2 GANGLIOSIDOSES
US10016514B2 (en) 2015-05-15 2018-07-10 New Hope Research Foundation Polynucleotides, vectors and methods for insertion and expression of transgenes
WO2019146745A1 (en) 2018-01-26 2019-08-01 国立大学法人徳島大学 Novel adeno-associated virus virion for treating tay-sachs disease and sandhoff disease

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008206428A (en) * 2007-02-24 2008-09-11 Univ Of Tokushima alpha-SUBUNIT COMPOSING beta-HEXOSAMINIDASE A (Hex A), Hex A HAVING THE SAME alpha-SUBUNIT, GENE, VECTOR AND TRANSFORMED CELL ENCODING THE SAME alpha-SUBUNIT

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008206428A (en) * 2007-02-24 2008-09-11 Univ Of Tokushima alpha-SUBUNIT COMPOSING beta-HEXOSAMINIDASE A (Hex A), Hex A HAVING THE SAME alpha-SUBUNIT, GENE, VECTOR AND TRANSFORMED CELL ENCODING THE SAME alpha-SUBUNIT

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KAZUHIKO MATSUOKA ET AL: "In silico Design ni Motozuku Kumikae Hito p-Hexosaminidase no Kokinoka", DAI 80 KAI NIPPON SEIKAGAKUKAI TAIKAI DAI 30 KAI THE MOLECULAR BIOLOGY SOCIETY OF JAPAN GODO TAIKAI KOEN YOSHISHU, 2007, pages 4T7-4 *
LEMIEUX M.J. ET AL: "Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis", J MOL BIOL., vol. 359, no. 4, 2006, pages 913 - 929 *
MARK B.L. ET AL: "Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease", J MOL BIOL., vol. 327, no. 5, 2003, pages 1093 - 1109 *
PENNYBACKER M. ET AL: "Identification of domains in human beta-hexosaminidase that determine substrate specificity", J BIOL CHEM., vol. 271, no. 29, 1996, pages 17377 - 17382 *
TSE R. ET AL: "Identification of an active acidic residue in the catalytic site of beta- hexosaminidase", BIOCHEMISTRY, vol. 35, no. 23, 1996, pages 7599 - 7607 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014061735A1 (en) 2012-10-19 2014-04-24 国立大学法人徳島大学 NOVEL HIGH-FUNCTIONING ENZYME HAVING ALTERED HUMAN β-HEXOSAMINIDASE B SUBSTRATE SPECIFICITY, AND HAVING PROTEASE RESISTANCE APPLIED THERETO
US20160137994A1 (en) * 2012-10-19 2016-05-19 Tokushima University NOVEL HIGH FUNCTIONAL ENZYME HAVING MODIFIED SUBSTRATE SPECIFICITY OF HUMAN ß-HEXOSAMINIDASE B AND EXHIBITING PROTEASE RESISTANCE
JPWO2014061735A1 (en) * 2012-10-19 2016-09-05 国立大学法人徳島大学 A novel high-functional enzyme that converts the substrate specificity of human β-hexosaminidase B and imparts protease resistance
US10400226B2 (en) 2012-10-19 2019-09-03 Tokushima University High functional enzyme having modified substrate specificity of human β-hexosaminidase B and exhibiting protease resistance
WO2015150922A3 (en) * 2014-03-17 2016-01-28 The Hospital For Sick Children β-HEXOSAMINIDASE PROTEIN VARIANTS AND ASSOCIATED METHODS FOR TREATING GM2 GANGLIOSIDOSES
US10400227B2 (en) 2014-03-17 2019-09-03 The University Of Manitoba β-hexosaminidase protein variants and associated methods for treating GM2 gangliosidoses
US10016514B2 (en) 2015-05-15 2018-07-10 New Hope Research Foundation Polynucleotides, vectors and methods for insertion and expression of transgenes
WO2019146745A1 (en) 2018-01-26 2019-08-01 国立大学法人徳島大学 Novel adeno-associated virus virion for treating tay-sachs disease and sandhoff disease
US12064460B2 (en) 2018-01-26 2024-08-20 Tokushima University Adeno-associated virus virion for treatment of Tay-Sachs disease and Sandhoff disease

Also Published As

Publication number Publication date
JP5678664B2 (en) 2015-03-04
JPWO2010082622A1 (en) 2012-07-05

Similar Documents

Publication Publication Date Title
JP6230158B2 (en) A novel high-functional enzyme that converts the substrate specificity of human β-hexosaminidase B and imparts protease resistance
JP5507242B2 (en) Pharmaceutical composition for enzyme replacement therapy
US20210284698A1 (en) Stabilized soluble pre-fusion rsv f polypeptides
JP5902679B2 (en) Tear lipocalin mutein that binds to IL-4Rα
JP4368925B2 (en) New high-performance enzyme with converted substrate specificity
CN112225793B (en) Lysosome targeting peptide, fusion protein thereof, adeno-associated virus vector carrying fusion protein coding sequence and application thereof
CN1924006A (en) Anti-glioma peptide of scorpion, preparation method and application thereof
JP5678664B2 (en) A novel high-performance enzyme that converts the substrate specificity of human β-hexosaminidase B
US9322008B2 (en) Mutants of L-asparaginase
US7666641B2 (en) Canine COX-2 proteins and uses thereof
US20090238813A1 (en) Compositions And Methods For Engineered Human Arginine Deiminases
US7335758B2 (en) Catalytic domain of ADAM33 and methods of use thereof
CN107868125A (en) MG53 mutant and its production and use
US7285408B2 (en) Crystalline form of an n231 mutant catalytic domain of ADAM33 and methods of use thereof
CA2506329C (en) Candida kefyr cytosine deaminase
WO2018230628A1 (en) Pharmaceutical combination for treatment of fabry disease and use thereof
CN117645661A (en) Polyethylene glycol modified IL-21 derivative and application thereof
TW202334195A (en) Recombinant factor viii variants
CN116949025A (en) Phenylalanine ammonia lyase mutant derived from candida nodosa and application thereof
CN118546903A (en) Enzyme capable of mediating large fragment DNA integration into mammalian genome and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10731291

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2010546654

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10731291

Country of ref document: EP

Kind code of ref document: A1