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 ( approximately 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl ( approximately 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.
Faro
J
J
Department of Immunology/Microbiology, Rush Medical College, Chicago, IL 60612, USA. jonathan_faro@rush.edu
Chen
Y
Y
Jhaveri
P
P
Oza
P
P
Spear
G T
GT
Lint
T F
TF
Gewurz
H
H
D000107:Acetylation; D037582:Complement Pathway, Mannose-Binding Lectin / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation / Q000276:immunology; D004792:Enzyme Precursors / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D008024:Ligands; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000302:isolation & purification; D000097604:Ficolins
D004792:Enzyme Precursors; D037102:Lectins; D008024:Ligands; D008077:Lipoproteins, LDL; D050606:Mannose-Binding Protein-Associated Serine Proteases; D000097604:Ficolins; C105296:MASP2 protein, human