l-Ficolin binding and lectin pathway activation by acetylated low-density lipoprotein

Summary l-Ficolin, like mannan-binding lectin (MBL), is a lectin pathway activator present in normal human plasma. Upon binding ligand, l-ficolin similarly initiates C4 cleavage via the serine protease MBL-associated serine protease-2 (MASP-2). We sought further insight into l-ficolin binding reactions and MASP-2activationbypassingplasmathroughGlcNAc-derivatizedSepharose. l-Ficolin bound in 1·0 M NaCl-ethylenediamine tetraacetic acid (EDTA), and remained bound in NaCl-free EDTA, while MASP-2 eluted in proenzyme form (~20% yield, > 40 000-fold purification). l-Ficolin was eluted with GlcNAc in 1·0 M NaCl (~10% yield, > 3000-fold purification), with trace amounts of C3, a2-macroglobulin and both native and activated MASP-2. These preparations were utilized to investigate l-ficolin reactivities with acetylated low-density lipoprotein (A-LDL) as a model ligand in albumin-free systems.l-Ficolin bound strongly to A-LDL in the absence as well as presence of calcium,includingsaline-EDTA,andwasoptimalin1·0 MNaCl-EDTA,but binding failed to occur in EDTA in the absence of NaCl. The addition of l-ficolin to immobilized A-LDL resulted in activation of MASP-2 in unmodifiedbutnotficolin-depletedplasmaunlessl-ficolinwasrestored.Weconclude that A-LDL is a useful ligand for investigation of l-ficolin function; both binding and activation are optimally examined in systems free of albumin; and ligand binding in 1·0 M NaCl in EDTA can be useful in the isolation of l-ficolin and native MASP-2.