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

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-2 activation by passing plasma through GlcNAc-derivatized Sepharose. 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 (similar to 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl (similar to 10% yield, > 3000-fold purification), with trace amounts of C3, alpha(2)-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, including saline-EDTA, and was optimal in 1.0 M NaCl-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 unmodified but not ficolin-depleted plasma unless L-ficolin was restored. We conclude 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.