Mannan-binding lectin (MBL), previously called ‘mannan-binding protein’ or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca2+-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.
Improvements on the purification of mannan-binding lectin and demonstration of its Ca2+-independent association with a C1s-like serine protease
- Views Icon Views
- PDF LinkPDF
- Share Icon Share
- Cite Icon Cite
Suet Mien TAN, Maxey C. M. CHUNG, Oi Lian KON, Steffen THIEL, Szu Hee LEE, Jinhua LU; Improvements on the purification of mannan-binding lectin and demonstration of its Ca2+-independent association with a C1s-like serine protease. Biochem J 15 October 1996; 319 (2): 329–332. doi: https://doi.org/10.1042/bj3190329
Download citation file: