Carbohydrate-binding specificities of mouse ficolin A, a splicing variant of ficolin A and ficolin B and their complex formation with MASP-2 and sMAP

Mannose-binding lectin (MBL) and ficolins are pattern recognition proteins acting in innate immunity, and they trigger the activation of the lectin complement pathway through MBL-associated serine proteases (MASPs). Upon activation of the lectin pathway, MASP-2 cleaves C4 and C2. A truncated form of MASP-2, named small MBL-associated protein (sMAP), is also associated with MBL/ficolin-MASP complexes. To clarify the role of sMAP, we have generated sMAP-deficient (sMAP−/−) mice by targeted disruption of the sMAP-specific exon. Because of the gene disruption, the expression level of MASP-2 was also decreased in sMAP−/− mice. When recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2, whereas the addition of rsMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP plays a regulatory role in the activation of the lectin pathway.

Section: Discussionmentioning

confidence: 97%

Small Mannose-Binding Lectin-Associated Protein Plays a Regulatory Role in the Lectin Complement Pathway

Iwaki

Kanno

Takahashi

et al. 2006

“…Recombinant FcnA (rFcnA) was prepared by using a Drosophila expression system with the pMT/Bip/V5-His A vector (Invitrogen, Carlsbad, CA) as previously described (25). Recombinant FcnB (rFcnB) was produced in the Chinese hamster ovary cells using a piggyBac transposon-mediated gene expression system: cotransfection with mouse FcnB cDNA-containing pIRCMV plasmid and pFerH plasmid.…”

Section: Recombinant Lectinsmentioning

confidence: 99%

“…C4-deposition assay was used to evaluate complement activation via the lectin pathway as previously described (25,27). In brief, mouse serum, mannose-eluate, or GlcNAc-eluate was incubated on a GlcNAc-BSAcoated microtiter plate in 100 ml TBS-Ca at 37˚C for 10 min.…”

Section: Complement Activation Assaymentioning

confidence: 99%

Mice Deficient in Ficolin, a Lectin Complement Pathway Recognition Molecule, Are Susceptible to Streptococcus pneumoniae Infection

Endo

Takahashi

Iwaki

et al. 2012

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)–deficient (Fcna−/−) and FcnA/ficolin B double-deficient (Fcna−/−b−/−) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna−/−, Fcnb−/−, and Fcna−/−b−/−) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna−/− but not in Fcna−/−b−/− mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

“…However, their varied locations in assorted tissues and species are suggestive of their differential functions. Notably, the tissue distribution and function of the human M-ficolin and its homologue (ficolin b) in pigs (23) and mice (24) are substantially different (25). Hitherto, no consensus has been reached on the subcellular localization and fate of M-ficolin in human monocytes, whether intracellular in secretory granules (19,20), on the cell surface (18), or secreted into the serum (26), although the localization of M-ficolin in the neutrophils was recently reported (19).…”

mentioning

confidence: 99%

Secreted M-Ficolin Anchors onto Monocyte Transmembrane G Protein-Coupled Receptor 43 and Cross Talks with Plasma C-Reactive Protein to Mediate Immune Signaling and Regulate Host Defense

Zhang

Yang

Ang³

et al. 2010

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin–GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin–GPCR43 complex. The collaboration among CRP–M-ficolin–GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin–CRP provides a powerful template for future design of potential immunomodulators.