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. 1996 Oct 15;319(Pt 2):329–332. doi: 10.1042/bj3190329

Improvements on the purification of mannan-binding lectin and demonstration of its Ca(2+)-independent association with a C1s-like serine protease.

S M Tan 1, M C Chung 1, O L Kon 1, S Thiel 1, S H Lee 1, J Lu 1
PMCID: PMC1217772  PMID: 8912663

Abstract

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 Ca(2+)-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.

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Selected References

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