# US 2010/0166728 A1 · Novel Highly Functional Enzyme Having Modified Substrate-Specificity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0166728 A1 - Document ID: US20100166728A1 - Status: Published application - Kind: A1 - Application number: 12/084,982 - Filed: 2006-11-17 - Published: 2010-07-01 - Earliest claimed: 2005-11-18 - Assignees: Tokyo Metropolitan Organization for Medical Research; Altif Laboratories - Inventors: Hitoshi Sakuraba; Youichi Tajima; Mai Ito; Seiichi Aikawa; Fumiko Aikawa - Web page: https://patentgrep.com/patent/US20100166728A1 ## CPC - C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)} - A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) - A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure - A61P 13/12: of the kidneys - A61P 17/00: Drugs for dermatological disorders - A61P 25/00: Drugs for disorders of the nervous system - A61P 27/02: Ophthalmic agents - A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)} - C12Y 302/01022: Alpha-galactosidase (3.2.1.22) - C12Y 302/01049: Alpha-N-acetylgalactosaminidase (3.2.1.49) - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12Y: ENZYMES - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present invention provides, as an enzyme which can be used for enzyme replacement therapy for Fabry disease, a protein having α-galactosidase activity, which shows no allergic adverse side effect, shows a high stability in blood, and can be easily incorporated into a cell of an affected organ. The protein of the present invention is a protein which has acquired α-galactosidase activity by changing the structure of the active site of wild-type human α-N-acetylgalactosaminidase. ## Claims ### Claim 1 A protein which has acquired α-galactosidase activity by changing the structure of the active site of wild-type human α-N-acetylgalactosaminidase. ### Claim 2 (depends on 1) The protein according to claim 1, wherein the protein has the substrate specificity of α-galactosidase. ### Claim 3 A protein having α-galactosidase activity, comprising an amino-acid sequence in which at least one of the 188th amino acid and the 191st amino acid in the amino-acid sequence of wild-type human α-N-acetylgalactosaminidase is replaced with another amino acid, or an amino-acid sequence in which one or several amino acids except the 188th amino acid and the 191st amino acid included in the replaced amino-acid sequence are deleted, replaced, or added. ### Claim 4 (depends on 3) The protein according to claim 3, wherein the 188th amino acid is replaced with glutamic acid or aspartic acid. ### Claim 5 (depends on 3) The protein according to claim 3, wherein the 191st amino acid is replaced with one selected from the group consisting of leucine, valine, isoleucine, phenylalanine, and methionine. ### Claim 6 (depends on 3) The protein according to claim 3, wherein the 188th amino acid is replaced with glutamic acid, and the 191st amino acid is replaced with leucine. ### Claim 7 A protein described by (a) or (b): (a) a protein containing any one of amino-acid sequences described by (i) to (iii): (i) an amino-acid sequence composed of the 18th amino acid to the 411th amino acid included in an amino-acid sequence in which the 188th amino acid is replaced with an amino acid other than serine in the amino-acid sequence shown in sequence No. 2; (ii) an amino-acid sequence composed of the 18th amino acid to the 411th amino acid included in an amino-acid sequence in which the 191st amino acid is replaced with an amino acid other than alanine in the amino-acid sequence shown in sequence No. 2; and (iii) an amino-acid sequence composed of the 18th amino acid to the 411th amino acid included in an amino-acid sequence in which the 188th amino acid is replaced with an amino acid other than serine and the 191st amino acid is replaced with an amino acid other than alanine in the amino-acid sequence shown in sequence No. 2; or (b) a protein containing an amino-acid sequence in which one or several amino acids other than the amino acid or amino acids located at the replacement site or sites are deleted, replaced, or added in any one of amino-acid sequences described by (i) to (iii), and having α-galactosidase activity. ### Claim 8 (depends on 7) A protein according to claim 7, wherein the amino acid other than serine is glutamic acid or aspartic acid. ### Claim 9 (depends on 7) The protein according to claim 7, wherein the amino acid other than alanine is one selected from the group consisting of leucine, valine, isoleucine, phenylalanine, and methionine. ### Claim 10 (depends on 7) The protein according to claim 7, wherein the amino acid other than serine is glutamic acid, and the amino acid other than alanine is leucine. ### Claim 11 (depends on 1) A gene encoding the protein according to claim 1. ### Claim 12 A gene comprising DNA described by (a) or (b): (a) DNA containing any one of base sequences described by (i) to (iii): (i) a base sequence composed of the 52nd to 1,236th bases included in a base sequence in which the 562nd to 564th bases are replaced with bases representing a codon of an amino acid other than serine in the base sequence shown in sequence No. 1; (ii) a base sequence composed of the 52nd to 1,236th bases included in a base sequence in which the 571st to 573rd bases are replaced with bases representing a codon of an amino acid other than alanine in the base sequence shown in sequence No. 1; and (iii) a base sequence composed of the 52nd to 1,236th bases included in a base sequence in which the 562nd to 564th bases are replaced with bases representing a codon of an amino acid other than serine and the 571st to 573rd bases are replaced with bases representing a codon of an amino acid other than alanine in the base sequence shown in sequence No. 1; or (b) DNA which encodes a protein having α-galactosidase activity and which hybridizes with DNA composed of a base sequence complementary to DNA containing any one of base sequences described by (i) to (iii) under a stringent condition, wherein bases corresponding to the bases at the replacement sites are identical to the bases at the replacement sites. ### Claim 13 (depends on 12) The gene according to claim 12, wherein the amino acid other than serine is glutamic acid or aspartic acid. ### Claim 14 (depends on 12) The gene according to claim 12, wherein the amino acid other than alanine is one selected from the group consisting of leucine, valine, isoleucine, phenylalanine, and methionine. ### Claim 15 (depends on 12) The gene according to claim 12, wherein the amino acid other than serine is glutamic acid, and the amino acid other than alanine is leucine. ### Claim 16 (depends on 11) A recombinant vector comprising the gene according to claim 11. ### Claim 17 (depends on 16) A transformant comprising the recombinant vector according to claim 16. ### Claim 18 A method of producing a protein having α-galactosidase activity, wherein the structure of the active site of wild-type human α-N-acetylgalactosaminidase is changed so that a substrate of α-galactosidase can be bound to the active site. ### Claim 19 (depends on 17) A method of producing a protein having α-galactosidase activity comprising a step of culturing the transformant according to claim 17, and a step of collecting the protein having α-galactosidase activity from the resulting cultured product. ### Claim 20 (depends on 1) A pharmaceutical composition comprising the protein according to claim 1. ### Claim 21 (depends on 20) A therapeutic agent for Fabry disease comprising the composition according to claim 20 as an active ingredient. ### Claim 22 (depends on 11) A pharmaceutical composition comprising the gene according to claim 11. ### Claim 23 (depends on 22) A gene therapeutic agent for Fabry disease comprising the composition according to claim 22 as an active ingredient. ### Claim 24 (depends on 20) A method of treating Fabry disease, wherein the pharmaceutical composition according to claim 20 is administered to a Fabry patient.