FCN1 (M-ficolin), which directly associates with immunoglobulin G1, is a molecular target of intravenous immunoglobulin therapy for Kawasaki disease

Kawasaki disease (KD), an acute systemic vasculitis of early childhood, is of unknown etiology. High-dose intravenous immunoglobulin (IVIG) is an effective treatment, but its molecular target remains elusive. DNA microarray analysis of peripheral blood mononuclear cells (PBMCs) revealed that at least 21 genes are drastically down-regulated after IVIG treatment in most KD patients. qRT-PCR analysis confirmed that the mRNA levels of five of these genes were considerably reduced in almost all KD patients after IVIG treatment. Western blot (Wb) of PBMC extracts revealed that levels of FCN1 (M-ficolin), a protein of the complement system that defends against infectious agents, were reduced after IVIG treatment in many KD patients. In another set of KD patients, Wb confirmed that levels of both FCN1 were greatly reduced after IVIG therapy. Wb revealed that the collagen-like domain of FCN1 directly bound to IgG1 in vitro through a portion of the CH1 and CH3 domains, and synthetic peptides corresponding to these domains of IgG1 efficiently inhibited these associations. These results suggest that FCN1 is a molecular target of intravenous IVIG in KD patients. We propose that these peptides and a humanized monoclonal antibody against FCN1 could be useful in combination therapy with IVIG.

Applied Biosystems, Foster City, CA) using the Assay-on-Demand TaqMan probe and genespecific primers. For HP, METTL7B, HPR, LOC100506229, FCN1, and FAP, assay kits (ID Hs00605928_g1, Hs00378551_m1, Hs00750565_s1, Hs01591421_m1, Hs00157572_m1, and Hs00990806_m1, respectively) were purchased from PE Applied Biosystems. The following oligonucleotides were used as primers and probes for GAPDH: GAPDH forward primer, 5'-CCATCAATGACCCCTTCATTG-3'; GAPDH reverse primer, 5'-TCTCGCTCCTGGAAGATGGT-3'; and GAPDH TaqMan probe, 5'-VIC-ACCTCAACTACATGGTTTAC-MGBNFQ-3'. Total RNA (500 ng) was reverse-transcribed using the High Capacity cDNA Archive Kit (ABI). The resultant cDNA was used as a template for PCR in a 20 μL reaction containing 10 µL of 2× Master Mix (TaKaRa, Otsu, Japan). PCR conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Each sample was assayed in quadruplicate, and the median threshold cycle (CT) values were used to calculate fold changes between the treated and control samples. A standard curve was generated from the amplification data for each primer using serial dilutions of PBMC RNA as the template. Fold change values were normalized to the corresponding levels of GAPDH levels using the standard curve method.
Plasmids. Double-stranded DNA of full human FCN1 cDNA was chemically synthesized by GenScript USA Inc. Human IgG1 cDNAs were purchased from New England Biolabs Inc. and Origene Inc. Plasmids encoding FCN1-N, FCN1-C, and dissected fragments of IgG1 were obtained by PCR-based amplification of these cDNAs and cloned into the AscI and NotI sites of mammalian expression vectors (pCMV6myc and p3FLAG) and a bacterial expression vector (pGST6P). All amplified sequences were confirmed by DNA sequencing. IP/Wb to examine the association between Flag-FCN1 and Myc-IgG1 proteins. Plasmid DNAs designed to express Flag-FCN1 or Myc-IgG1 proteins under the control of the cytomegalovirus (CMV) promoter were transfected to 293T cells using Lipofectamine (Thermo Fisher Scientific). After 48 h incubation in DMEM, each cell extract (dissolved in 500 µL of TNE250 including protease inhibitors: TNEI) was mixed with anti-Myc antibody, and then subjected to inversion mixing process on a rotator overnight at 4°C to facilitate complete binding of FCN-N and IgG1 proteins. After brief centrifugation (4°C; 4,900 × g; 2 min), the precipitate was rinsed again three times with 500 µL TNEI. To the final precipitate, 15 µL of 2× SDS-PAGE sample buffer was added; the sample was boiled in hot water for 7 min, and then subjected to Wb using anti-Myc antibody (IP/Wb) or anti-FLAG antibody (loading control).

Pull-down experiments using GST-FCN1-N.
Plasmid DNA designed to express GST-FCN1-N fragment was cloned into the AscI and NotI sites of GST-fused protein expression vector (pGST6P) 23 derived from pGEX6P (Amersham Pharmacia), and then transfected into E. coli (BL21 RIL) using competent cells prepared according to the SEM protocol 24 . Single colonies that expressed GST-FCN1-N proteins with the highest efficiency were selected. Next, glutathione-Sepharose beads (15 µL) used for affinity purification were mixed with cell extracts from 293T cells (10 µg) expressing Myc-IgG1 fragments and then subjected to inversion mixing on a rotator overnight at 4°C to facilitate complete binding of GST-FCN-N and IgG1 proteins. After brief centrifugation (4°C; 4,900 × g; 2 min), the precipitate was rinsed again three times with 500 µL TNEI. To the final precipitate, 15 µL 2× SDS-PAGE sample buffer was added; the sample was boiled in hot water for 7 min, and then subjected to Wb using anti-Myc antibody (IP/Wb) or anti-FLAG antibody (loading control).

Inhibition of FCN-N and IgG1 fragments by synthetic peptides. Glutathione-
Sepharose beads (15 µL) containing affinity purified GST-FCN1-N proteins were rinsed twice with TNE, and the precipitate was dissolved in 500 µL TNEI. Next, chemically synthesized peptides were added, and the samples were subjected to inversion mixing on a rotator for 1 h at 4°C. To this mixture was added 10 µg cell extract from 293T cells expressing Myc-IgG1 fragments, and the sample was again subjected to inversion mixing for 1 h at 4°C to allow competitive binding of peptides and Myc-IgG1-3d or Myc-IgG1-CH1p fragments.
After brief centrifugation (4°C; 4,900 × g; 2 min), the precipitate was rinsed again three times with 500 µL TNEI. To the final precipitate, 15 µL of 2× SDS-PAGE sample buffer was added; the sample was boiled in hot water for 7 min, and then subjected to Wb using anti-Myc antibody (IP/Wb) or anti-FLAG antibody (loading control).     , and HPR (f) were determined by qRT-PCR using using purified RNA from each KD patient on the indicated day. Day 1 (d1) indicates blood collected before IVIG treatment; d2 or d7 indicates blood collected 2-3 days or 6-8 days after IVIG treatment, respectively. Horizontal bar in each dot plot denotes average value of 19 KD patients, with standard deviation bars. The vertical axis indicates mRNA level (arbitrary units, a.u.) relative to that at 1d, which was fixed at 1.0 a.u. Average and standard deviation values (error bars) are shown. , and HPR (f) were determined by qRT-PCR using purified RNA from HVs (leftmost column) and from each KD patient (lower x-axis labels) on the indicated day. Number for each bar (Day) indicates the date when blood was collected from each patient. The vertical axis indicates mRNA level (a.u.) relative to that of HV, which was fixed at 1.0 a.u.