WO2001055198A9 - Haut- und magen-spezifischer sphingolipid-aktivator - Google Patents
Haut- und magen-spezifischer sphingolipid-aktivatorInfo
- Publication number
- WO2001055198A9 WO2001055198A9 PCT/EP2001/000877 EP0100877W WO0155198A9 WO 2001055198 A9 WO2001055198 A9 WO 2001055198A9 EP 0100877 W EP0100877 W EP 0100877W WO 0155198 A9 WO0155198 A9 WO 0155198A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- saposin
- seq
- syndrome
- pro
- esap
- Prior art date
Links
- 239000012190 activator Substances 0.000 title claims description 6
- 150000003408 sphingolipids Chemical class 0.000 title claims description 6
- 102100036197 Prosaposin Human genes 0.000 claims abstract description 15
- 101710152403 Prosaposin Proteins 0.000 claims abstract description 15
- 239000012634 fragment Substances 0.000 claims description 17
- 208000011580 syndromic disease Diseases 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 210000002784 stomach Anatomy 0.000 claims description 6
- 239000000032 diagnostic agent Substances 0.000 claims description 4
- 229940039227 diagnostic agent Drugs 0.000 claims description 4
- 208000010557 Lipid storage disease Diseases 0.000 claims description 3
- 239000002537 cosmetic Substances 0.000 claims description 3
- 230000007547 defect Effects 0.000 claims description 3
- 230000037356 lipid metabolism Effects 0.000 claims description 3
- 208000014416 lysosomal lipid storage disease Diseases 0.000 claims description 3
- 230000002018 overexpression Effects 0.000 claims description 3
- 241000238097 Callinectes sapidus Species 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- 201000009053 Neurodermatitis Diseases 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000012790 confirmation Methods 0.000 claims description 2
- 230000007812 deficiency Effects 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 230000004137 sphingolipid metabolism Effects 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims 2
- 201000011442 Metachromatic leukodystrophy Diseases 0.000 claims 1
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 3
- 230000008827 biological function Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 101150024973 PNPLA2 gene Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000017852 Saposin Human genes 0.000 description 1
- 108050007079 Saposin Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- -1 aromatic amino acids Chemical class 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003458 metachromatic effect Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- 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 sphingolipid activator.
- Lysosomal enzymes of lipid metabolism often require specific activator proteins.
- the lysosomal sphingolipid activators Sap-A, Sap-B, Sap-C and Sap-D are generated by proteolytic cleavage from a precursor, the so-called pro-saposin.
- the individual saposins activate various enzymes that break down sphingolipids. Their mode of operation is based on the fact that they present the degradable lipids to the enzymes or make them more easily accessible.
- a skin-specific pro-saposin with the sequence (Seq. ID No. 5):
- the protein according to the invention also includes proteins which are formed by modification in the side chains and which have the biological effectiveness of eSAP. sen, especially phosphorylations, glycolizations, amidations and other derivatizations in the side chains. It is also clear to the person skilled in the art that, by modifications in the primary sequence, fragments can be produced which likewise ensure the biological function of the protein according to the invention. These include, for example, fragmentations, where biologically active fragments can easily be determined by a person skilled in the art by means of appropriate experiments. These include, for example, site directed mutagenesis, with the aid of which structural variants which also have biological activity can be produced. In addition, protein chemistry can also be used to exchange amino acids or sequences or motifs within the primary structure.
- exchanges are well known to those skilled in the art and include, for example, conservative exchanges in which, for example, serine is exchanged for threonine.
- Other groups known to those skilled in the art are of course also suitable as exchange candidates.
- the groups of non-polar amino acids, polar amino acids, aromatic amino acids can be exchanged with one another without profound changes in the biological function being expected.
- the figure shows the result of the Northern blot analysis.
- a comparison of partial images A and B shows that a strong eSAP signal at 2.5 kB is only observed in the skin and stomach from a large number of human tissues. The weaker and shorter signals in the heart and muscle do not correspond to a functional transcript.
- Fragments which represent sphingolipid activators are also claimed according to the invention. These preferably have a length of 40 to 100, preferably 75 to 85, amino acids.
- Preferred fragments have the sequences mentioned in the sequence listing (Seq. ID No. 1 to 4):
- the polypeptide Seq. ID No. 1 becomes eSAP-A (position 61-144), the polypeptide
- BEST ⁇ T.GUNGS OP ⁇ E Seq. ID No. 2 becomes eSAP-B (position 181-257), the polypeptide Seq. ID No. 3 is eSAP-C (positions 290-369) and the polypeptide Seq. ID No. 4 is called e-SAP-D (position 392 - 473).
- the invention relates to pro-saposin and its fragments also encode nucleic acids of the invention for the purpose and complementary nucleic acids, which can for example be used as antisense Oligonucieotide.
- a pharmaceutical, cosmetic or diagnostic agent containing a skin-specific pro-saposin according to claim 1, a fragment according to claim 2 or nucleic acids according to one of claims 3 or 4 together with customary auxiliaries.
- Another aspect is the use of the medicinal or diagnostic agent according to claim 5 for the therapy or diagnosis of diseases related to lipid metabolism, in particular to the sphingolipid metabolism and lipid storage diseases, in particular neurodermatitis.
- saposin according to the invention or its fragments can be used as cosmetic agents in formulations known to those skilled in the art.
- the invention also relates to a transgenic animal with overexpression (gain of function) or gene deficiency or gene defect (loss of function) for a pro-saposin or fragment according to one of claims 1 and / or 2, and a cell line which is characterized in that that it overexpresses a pro-saposin or a fragment according to one of claims 1 and / or 2.
- overexpression gain of function
- gene deficiency loss of function
- the heSAP cDNA was amplified using PCR.
- the amplified cDNA was subcloned and the sequence subsequently verified.
- the tissue distribution of the heSAP was analyzed using the Norhern blot technique.
- purchased blots were used (OriGene Technologie Inc., Catalog #: HB-2010, HB-1102).
- the 318bp PstI fragment of the cDNA was used as a probe.
- the cDNA fragment was radiolabelled.
- the hybridization took place via Nadht at 65 ° C.
- the radioactive signals were measured using the Cyclone Storage Phosphor System (both from Packard Instrument Company, Dreieich) and evaluated using the OptiQuant program.
- the blots were normalized with ß-actin.
- the entire cDNA was cloned into a eukaryotic expression vector under the control of the CMV promoter.
- HEK293 cells were electroporated with the construct and subsequently selected with G418. Cell clones that survived the selection were examined for heSAP overexpression.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Biophysics (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2001233715A AU2001233715A1 (en) | 2000-01-26 | 2001-01-26 | Dermo- and gastro-specific sphingolipid activator |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10003154.4 | 2000-01-26 | ||
DE10003154 | 2000-01-26 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2001055198A2 WO2001055198A2 (de) | 2001-08-02 |
WO2001055198A3 WO2001055198A3 (de) | 2002-03-14 |
WO2001055198A9 true WO2001055198A9 (de) | 2003-08-14 |
Family
ID=7628688
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/000877 WO2001055198A2 (de) | 2000-01-26 | 2001-01-26 | Haut- und magen-spezifischer sphingolipid-aktivator |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2001233715A1 (de) |
WO (1) | WO2001055198A2 (de) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001034627A1 (en) * | 1999-11-12 | 2001-05-17 | Human Genome Sciences, Inc. | 28 human secreted proteins |
DK1605965T3 (da) * | 2003-03-26 | 2013-04-15 | Evotec Internat Gmbh | Anvendelse af saposin-beslægtede proteiner til forebyggelse og behandling af obesitet, diabetes og/eller metabolisk syndrom |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5700909A (en) * | 1993-07-30 | 1997-12-23 | The Regents Of The University Of California | Prosaposin and cytokine-derived peptides |
JP2001515866A (ja) * | 1997-09-09 | 2001-09-25 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | プロサポシン受容体アゴニストを利用したアポトーシスの阻害 |
AU772778B2 (en) * | 1998-07-13 | 2004-05-06 | University Of Southern California | Novel inhibitors of angiogenesis and tumor growth |
WO2000005361A1 (en) * | 1998-07-23 | 2000-02-03 | Smithkline Beecham P.L.C. | Human sbpsapl gene with homology to the prosaposin family of neurotrophic factors |
WO2000055632A1 (en) * | 1999-03-17 | 2000-09-21 | Women's And Children's Hospital | Diagnosis of lysosomal storage disorders using saposins and other markers |
FR2797402B1 (fr) * | 1999-07-15 | 2004-03-12 | Biomerieux Stelhys | Utilisation d'un polypeptide pour detecter, prevenir ou traiter un etat pathologique associe a une maladie degenerative, neurologique ou autoimmune |
-
2001
- 2001-01-26 WO PCT/EP2001/000877 patent/WO2001055198A2/de active Application Filing
- 2001-01-26 AU AU2001233715A patent/AU2001233715A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2001055198A2 (de) | 2001-08-02 |
AU2001233715A1 (en) | 2001-08-07 |
WO2001055198A3 (de) | 2002-03-14 |
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