Masp-2, a complement-fixing enzyme, and uses for it

A technology of MASP-2, application, applied in the field of MASP-2, complement fixation enzyme and application thereof

Inactive Publication Date: 2003-10-15
詹斯·C·詹斯尼厄斯 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, the overall survival of the population may benefit from a wide range of individual MBL concentrations

Method used

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  • Masp-2, a complement-fixing enzyme, and uses for it
  • Masp-2, a complement-fixing enzyme, and uses for it
  • Masp-2, a complement-fixing enzyme, and uses for it

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] Example 1: Identification of MASP-2

[0233] Human plasma proteins were purified by affinity chromatography on mannan- and N-acetylglucosamine-derivatized sepharose beads and combined with carbohydrates in a calcium-dependent manner (i.e., lectins and lectin-related proteins). Compounds bound to protein complexes. Pooled CPD-plasma (2.5 l) diluted with buffer containing EDTA and enzyme inhibitors was run through Sepharose 2B CL and Mannan Sepharose. Add coagulation inhibitor PPACK (D-phenylalanyl-prolyl-arginyl-chloromethyl ketone) and CaCl 2 . The samples were passed through Sepharose 2B-CL and Mannan-Sepharose, and the calcium-dependently bound proteins to Mannan-Sepharose were eluted with buffer containing EDTA. The recalcified eluate was passed through a GIcNAc-Sepharose column and eluted as described above to yield 20 ml of "lectin preparation".

[0234] The protein preparation was analyzed by SDS-PAGE and blot on PVDF-membrane. Blot with chicken antibody agai...

Embodiment 2

[0236] Example 2: Preparation of antibodies against mannan-binding lectin-related serine proteases

[0237] According to C. Koch, The State Serum Institute, Copenhagen, animals initially immunized with BCG (Bacillus Calmette Guerin vaccine) were immunized with a synthetic peptide conjugated to PPD (Purified Protein Derivative of Tuberculin). Antibodies called anti-N'MASP-1, anti-C'MASP-1, and anti-N'MASP-2 were derived from antibodies corresponding to the first 19 amino acid residues of MASP-1 and the last 19 amino acid residues of MASP-1, respectively. Residues and the first 19 amino acid residues of MASP-2 in immunized rabbits. Chicken anti-C'MASP-2 antibodies were obtained from chickens immunized with a mixture of two peptides (residues 505 to 523 and 538 to 556) representing the C-terminal partial sequence of MASP-2. All peptides have an additional C-terminal cysteine ​​for coupling. Antibody and normal chicken IgG26 were purified from egg yolk.

[0238] Purification of...

Embodiment 3

[0239] Example 3: Amino acid sequences of 52 kDa and 20 kDa polypeptides. The lectin preparation was concentrated, subjected to SDS-PAGE electrophoresis, and transferred to a PVDF-membrane. Two bands were revealed with anti-bovine agglutinin antibody 25 . Stain the rest of the blot with Coomassie brilliant blue. The band corresponding to the immuno-stained 52 kDa band (determined to represent approximately 5% of the Coomassie-stained protein) was excised and subjected to sequence analysis on an Applied Biosystems protein sequencer. After the anti-N'MASP-2 antibody was raised, a similar western blot was done using the anti-N'MASP-2 antibody. The 20 kDa and 52 kDa bands at the amino terminus of the protein observed with this antibody were subjected to sequence analysis. Peptides were prepared by trypsinizing proteins from two bands from another blot, fractionated by reverse phase chromatography, and the major peak peptide was subjected to sequence analysis.

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PUM

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Abstract

The present invention relates to substantially pure mannan-binding lectin associated serine protease-2 (MASP-2) polypeptides and fragments thereof as well as nucleic acids encoding such polpeptides. Furthermore, the present invention relates to uses of a substantially pure polypeptide comprising amino acid sequences derived from mannan-binding lectin associated serine protease-2 (MASP2) or a functional homologue thereof for the production of a pharmaceutical composition as well as pharmaceutical compositions comprising MASP-2 and / or MASP-2 fragments. In addition the present invention relates to inhibitors of MASP-2 and pharmaceutical compositions comparing such inhibitors. Methods for detecting MASP-2 nucleic acid expression are included in the invention.

Description

field of invention [0001] The present invention is in the general field involving the intrinsic pathway of complement fixation by mannan-binding lectin (MBL, also known as mannan-binding protein). Background of the invention [0002] The complement system includes a complex series of enzymes and non-enzymatic proteins important for natural function and adaptive immune defense 1 . Two modes of activation were not known until recently, the classical pathway initiated by antibody-antigen complexes and the alternative pathway initiated by certain structures on the bacterial surface. A third and novel antibody-independent pathway of complement activation has been described 2 . This pathway is initiated when mannan-binding lectin (MBL, originally described as mannan-binding protein, MBP, see Ezekowitz, US Patent 5,270,199) binds carbohydrates. [0003] MBL is structurally related to the C1q subcomponent of the complement component C1, and it appears t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53A61K31/7052A61K38/00A61K38/48A61K39/395A61K45/00A61K48/00A61P31/00C07K14/47C07K16/18C12NC12N9/64C12N15/09C12N15/12C12N15/57C12Q1/37C12Q1/68G01N33/566G01N33/573
CPCG01N33/573C07K2319/42G01N2333/96433A61K38/482C07K2319/21C12N9/6424C07K16/40C12Y304/21104A61P31/00A61P31/12A61P31/14A61P31/18
Inventor 詹斯·C·詹斯尼厄斯斯蒂芬·蒂尔
Owner 詹斯·C·詹斯尼厄斯
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