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CN-121975757-A - Urate oxidase variant and glycosylation modified form thereof

CN121975757ACN 121975757 ACN121975757 ACN 121975757ACN-121975757-A

Abstract

The invention relates to the technical field of protein medicines, in particular to a urate oxidase variant and a glycosylation modification form thereof. According to the invention, the engineering N-glycosylation site is introduced into the urate oxidase, and a pair of engineering disulfide bonds are additionally introduced into a tetramer interface, so that the obtained glycosylation modified urate oxidase variant can improve folding stability, in-vivo expression and immunological properties on the basis of keeping enzyme activity.

Inventors

  • YAN CHUANGYE
  • XIAO YUAN
  • LI JIACHEN
  • LIU YIDONG

Assignees

  • 清华大学

Dates

Publication Date
20260505
Application Date
20260204

Claims (15)

  1. 1. A variant of urate oxidase which is mutated at least one site between positions 40 to 50, 210 to 220 or 235 to 245 in the amino acid sequence of wild type urate oxidase to form an N-glycosylation site; the N-glycosylation site is NXS or NXT, wherein X is any amino acid which is not proline.
  2. 2. The variant according to claim 1, wherein the mutation comprises a substitution, deletion and/or addition of an amino acid, and the N-glycosylation site is NTS, NVT or NKTS.
  3. 3. The variant according to claim 2, wherein the mutation comprises at least one of the following: inserting or substituting at least one amino acid residue at any position between 40 and 50 positions to enable the amino acid residue to contain NTS; inserting or substituting at least one amino acid residue at any position between 210 and 220 to ensure that the amino acid residue contains NVT; at least one amino acid residue is inserted or substituted at any position between 235-245 so as to contain NKTS.
  4. 4. A variant according to claim 3, characterized in that the mutation is: E at position 44 to N; and/or E at position 214 to N; And/or the ARQ mutation at 241-243 th site is NKTS.
  5. 5. The variant according to any of claims 1 to 4, wherein the mutation further comprises a mutation in at least one position between positions 280-290 in the amino acid sequence of the wild-type urate oxidase to contain a cysteine.
  6. 6. The variant according to claim 5, wherein the mutation further comprises a mutation at positions 283-284 SD to CC in the amino acid sequence of wild-type urate oxidase.
  7. 7. The variant according to any one of claims 1 to 6, wherein the wild-type urate oxidase is a urate oxidase derived from aspergillus flavus, preferably having the amino acid sequence shown in SEQ ID No. 1.
  8. 8. The variant according to any of claims 1 to 7, further comprising a signal peptide and/or a purification tag at the N-terminus, The signal peptide is at least one of a Mouse IgG kappa signal peptide, an IL-2 signal peptide, an INS signal peptide, an SPN signal peptide and a CTRB2 signal peptide; the purification tag is at least one of a 6 xHis tag, an 8 xHis tag, a 10 xHis tag, a Strep tag, an HA tag, a GST tag, an MBP tag, a GFP tag, a Flag tag and a AlFA tag.
  9. 9. Biological material comprising at least one of: a) A nucleic acid encoding the variant of any one of claims 1-8; b) An expression unit comprising a promoter and the nucleic acid of a); c) An expression vector comprising a) said nucleic acid and/or b) said expression unit; d) A transformant comprising the plasmid vector of c), or the genome thereof, having the nucleic acid of a) and/or the expression unit of b) integrated therein.
  10. 10. A glycosylation modified urate oxidase variant, which is a variant according to any one of claims 1 to 8 wherein the N residue in the N-glycosylation site is glycosylated.
  11. 11. The glycosylation modified urate oxidase variant according to claim 10, wherein the glycosylation comprises a reducing end of the sugar chain linked to the amide nitrogen atom of the N residue via an N-glycosidic bond.
  12. 12. The glycosylation modified urate oxidase variant according to claim 10 or 11, wherein it is tetrameric and disulfide bonds are formed between tetrameric subunits via cysteines.
  13. 13. A method of preparing a glycosylation modified urate oxidase variant according to any one of claims 10 to 12, comprising culturing the transformant according to claim 9 to obtain the variant according to any one of claims 1 to 8, chemically modifying, and linking an N-sugar chain to an amide nitrogen atom of an N residue; or culturing the transformant according to claim 9, to obtain a culture product containing the glycosylation-modified urate oxidase variant.
  14. 14. Use of the variant according to any one of claims 1 to 8, the glycosylation-modified urate oxidase variant according to any one of claims 10 to 12 or the culture product produced according to claim 13 for the preparation of a medicament for the prophylaxis and treatment of hyperuricemia-related diseases.
  15. 15. A medicament for preventing and treating hyperuricemia-related diseases, which comprises the variant of any one of claims 1 to 8, the glycosylation-modified urate oxidase variant of any one of claims 10 to 12 or the culture product produced by claim 13.

Description

Urate oxidase variant and glycosylation modified form thereof Technical Field The invention relates to the technical field of protein medicines, in particular to a urate oxidase variant and a glycosylation modification form thereof. Background Uricase (Uricase, EC 1.7.3.3), also known as uricase, is a key enzyme class capable of catalyzing oxidative decomposition of uric acid, and is widely found in various organisms in nature, such as microorganisms (bacteria, fungi), insects, and parts of mammals. However, the functional urate oxidase gene is naturally lacking in human body, and the enzyme cannot be autonomously synthesized. The uric acid oxidase has the core action mechanism that uric acid (2, 6, 8-trihydroxy purine) is specifically oxidized and decomposed into allantoin, carbon dioxide and hydrogen peroxide in the presence of oxygen, wherein the water solubility of the allantoin is more than 50 times of that of uric acid, and the allantoin is easier to excrete through kidneys, so that the in-vivo uric acid level is rapidly reduced. The clinical application of the urate oxidase mainly comprises the following aspects of rapidly reducing uric acid and preventing kidney injury in emergency treatment of acute hyperuricemia caused by tumor lysis syndrome and the like, taking the urate oxidase as a first-line emergency medicine, serving as a substitute treatment for chronic gout refractory treatment, maintaining stable blood uric acid, and assisting in diagnosis of liver diseases, metabolic syndrome and the like by detecting serum activity of the urate oxidase. Although urate oxidase has remarkable effect in clinical application, the natural urate oxidase or semisynthetic urate oxidase commonly used at present still has a plurality of defects: the immunogenicity is strong, the clinical urate oxidase is mostly derived from microorganisms or animal tissues, the amino acid sequence and the human protein have large difference, and the immune response is easy to be induced in vivo. The thermal stability is poor, the spatial structure of the natural urate oxidase is easily affected by temperature, and the activity can be lost when the natural urate oxidase is placed at normal temperature. The clinical needs low-temperature refrigeration, the whole-course cold chain is needed for transportation, the cost and the operation difficulty are increased, the in-vivo temperature can accelerate the inactivation of the cold chain, the action time is shortened, and the dosage or the frequency of administration is increased. The high temperature tolerance is insufficient, uric acid oxidase can be rapidly deactivated in medical environments in tropical areas, basic medical institutions lacking cold chain equipment and other high temperature environments, and in the preparation of medicines, part of process temperature conditions can increase difficulty due to high temperature instability of uric acid oxidase and influence the quality stability of products. Therefore, by means of technical means such as genetic engineering, protein engineering and the like, the uric acid oxidase is structurally modified, so that the immunogenicity of the uric acid oxidase is reduced, the thermal stability and the high-temperature tolerance of the uric acid oxidase are improved, the enzyme activity is enhanced, the in vivo half-life is prolonged, and the uric acid oxidase becomes a research hot spot in the current biopharmaceutical field, and is also a key direction for promoting the upgrading and updating of uric acid oxidase drugs and improving the treatment experience of patients. Disclosure of Invention In view of the above, the technical problem underlying the present invention is to provide urate oxidase variants and glycosylation modified forms thereof. The invention provides a variant of urate oxidase, which is mutated at least one site between the 40 th site and the 50 th site, between the 210 th site and the 220 th site or between the 235 th site and the 245 th site in the amino acid sequence of wild urate oxidase to form an N-glycosylation site; the N-glycosylation site is NXS or NXT, wherein X is any amino acid which is not proline. The invention designs and screens glycosylation sites by taking Aspergillus flavus urate oxidase (Aspergillus flavus uricase, afUox for short, uniprot ID:Q 00511) as a model protein. Based on AfUox's three-dimensional structure and functional region analysis, engineering regions with hydrophilic surfaces and minimal impact on protein folding, polymerization, and function are screened away from enzyme active sites, multimeric interfaces, hydrophobic cores, and key interaction surfaces. In order to further improve the structural stability and in vivo tolerance of AfUox tetramers, disulfide bonds are introduced at the tetramer interface of uricase, so that the tetramer structure is more stable and is not easy to depolymerize into dimers or monomers. The glycosylation modified urate oxidase variant prepared by t