Masp-2, a complement-fixing enzyme, and uses for it
View Patent ↗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.
1. A method of assaying for the immune defense complement pathway activating activity of a mature MASP-2 polypeptide in a complex with mannan-binding lectin (MEL) wherein the mature MASP-2 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:2 from position 16 through position 686, the method comprising:
(i) obtaining a sample comprising MBL/MASP-2 complexes,
(ii) contacting said sample with at least one MASP-2 substrate in a solution having a salt concentration greater than 0.2M, and,
(iii) detecting the amount of MASP-2 substrate cleaved, whereby the MBL/MASP-2 complex complement pathway activating activity is quantified.
2. The method according to claim 1 wherein said method further comprises the steps of:
(a) applying the sample of step (i) to a solid phase substrate to obtain bound complexes,
(b) performing steps (ii) and (iii), and
(c) correlating the amount of cleaved MASP-2 substrate to the amount of MASP-2 in the sample of step (i),
whereby the activity of MASP-2 in the MBL/MASP-2 complexes is determined.
3. The method according to claim 2 , wherein the solid phase is a mannan coating.
4. The method according to claim 1 , wherein the at least one MASP-2 substrate is a complement factor cleavable by the MBL/MASP-2 complex.
5. The method according to claim 4 wherein the at least one MASP-2 substrate is selected from the group consisting of C2, C3, and C4.
6. The method according to claim 2 , wherein the cleaved complement factor is detected by means of antibodies directed to the complement factor.
7. The method according to claim 1 wherein the salt is selected from the group consisting of NaCl, KCl, MgCl 2 , CaCl 2 , NaI, KCl, MgI 2 , CaI 2 , NaBr, KBr, MgBr 2 , CaBr 2 , Na 2 S 2 O 3 , (NH 4 ) 2 SO 4 , and (NH 4 )HCO 3 .
8. The method of claim 1 wherein the salt concentration is in the range of 0.5M to 5.0M.
9. The method of claim 1 wherein the sample is a biological sample.