title: A novel measurement method for activation of the lectin complement pathway via both mannose-binding lectin (MBL) and L-ficolin.
abstract: 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.
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inoshita
authors.forename: Misao
authors.initials: M
authors.lastname: Matsushita
authors.forename: Shunichi
authors.initials: S
authors.lastname: Koide
authors.forename: Gaku
authors.initials: G
authors.lastname: Kusaba
authors.forename: Masaya
authors.initials: M
authors.lastname: Ishii
authors.forename: Kisara
authors.initials: K
authors.lastname: Onda
authors.forename: Min Jin
authors.initials: MJ
authors.lastname: Gi
authors.forename: Munehiro
authors.initials: M
authors.lastname: Nakata
authors.forename: Isao
authors.initials: I
authors.lastname: Ohsawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Horikoshi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tomino
mesh_terms: 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
chemical_list: D000117:Acetylglucosamine; D003181:Complement C4; D037102:Lectins; D037601:Mannose-Binding Lectin; D010714:Phosphatidylethanolamines; D000097604:Ficolins; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine