A novel measurement method for activation of the lectin complement pathway via both mannose-binding lectin (MBL) and L-ficolin.
Mannose-binding lectin (MBL), L-ficolin and H-ficolin are human serum lectins, all of which form complexes with MBL-associated serine proteases (MASP). The lectin-MASP complexes bind to the surface of microbes, leading to activation of the lectin pathway of complement. Enzyme-linked immunosorbent assays (ELISA) of the lectin pathway activity reported so far determined the activity via either MBL or L-ficolin, but an assay of activity via plural host defense lectins has not been established. To measure the lectin pathway activation mediated by plural lectins simultaneously, we developed an ELISA system in which N-acetylglucosamine-pentamer conjugated to dipalmitoylphosphatidylethanolamine (GN5-DPPE) was employed as a ligand for the lectins. In our ELISA system, both purified MBL and L-ficolin isolated from serum diluted in a buffer containing high ionic NaCl bound to GN5-DPPE and activated C4. Purified H-ficolin was not capable of binding to GN5-DPPE. MBL and L-ficolin in MBL-sufficient serum also bound to GN5-DPPE and activated C4. Mannose and N-acetylgalactosamine inhibited binding of MBL and L-ficolin to GN5-DPPE, respectively. MBL-deficient serum that had been depleted of L-ficolin did not exhibit C4 activation, but addition of both or either purified MBL and/or L-ficolin to the serum restored the activation in a dose-dependent manner. Thus, C4 cleaving activity could be evaluated with the co-existence of MBL and L-ficolin in vitro. In conclusion, we propose a novel method using GN5-DPPE for investigating the MBL- and L-ficolin-dependent lectin pathway and anticipate that this method will be useful in innate immunity and clinical research.
Inoshita
Hiroyuki
H
Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Matsushita
Misao
M
Koide
Shunichi
S
Kusaba
Gaku
G
Ishii
Masaya
M
Onda
Kisara
K
Gi
Min Jin
MJ
Nakata
Munehiro
M
Ohsawa
Isao
I
Horikoshi
Satoshi
S
Ohi
Hiroyuki
H
Tomino
Yasuhiko
Y
D000117:Acetylglucosamine / Q000737:chemistry / Q000276:immunology; D001667:Binding, Competitive; D003167:Complement Activation / Q000276:immunology*; D003181:Complement C4 / Q000737:chemistry / Q000276:immunology; D037582:Complement Pathway, Mannose-Binding Lectin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000276:immunology*; D037601:Mannose-Binding Lectin / Q000737:chemistry / Q000276:immunology*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000276:immunology; D000097604:Ficolins
D000117:Acetylglucosamine; D003181:Complement C4; D037102:Lectins; D037601:Mannose-Binding Lectin; D010714:Phosphatidylethanolamines; D000097604:Ficolins; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine