Improvements on the purification of mannan-binding lectin and demonstration of its Ca(2+)-independent association with a C1s-like serine protease.
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.
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore.
S M
SM
Tan
M C
MC
Chung
O L
OL
Kon
S
S
Thiel
S H
SH
Lee
J
J
Lu
D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D037222:Collectins; D006801:Humans; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D002352:Carrier Proteins; D037222:Collectins; D037102:Lectins; D012697:Serine Endopeptidases; D002118:Calcium