Functional characterization of a ficolin-mediated complement pathway in amphioxus

Abstract

The ficolin-mediated complement pathway plays an important role in vertebrate immunity, but it is not clear whether this pathway exists in invertebrates. Here we identified homologs of ficolin pathway components from the cephalochordate amphioxus and investigated whether they had been co-opted into a functional ficolin pathway. Four of these homologs, ficolin FCN1, serine protease MASP1 and MASP3, and complement component C3, were highly expressed in mucosal tissues and gonads, and were significantly up-regulated following bacterial infection. Recombinant FCN1 could induce hemagglutination, discriminate among sugar components, and specifically recognize and aggregate several bacteria (especially gram-positive strains) without showing bactericidal activity. This suggested that FCN1 is a dedicated pattern-recognition receptor. Recombinant serine protease MASP1/3 formed complexes with recombinant FCN1 and facilitated the activation of native C3 protein in amphioxus humoral fluid, in which C3 acted as an immune effector. We conclude that amphioxus have developed a functional ficolin-complement pathway. Because ficolin pathway components have not been reported in non-chordate species, our findings supported the idea that this pathway may represent a chordate-specific innovation in the evolution of the complement system.

FIGURE 1.

A, sequence alignment of ficolins from various species. B, architecture of cephalochordate amphioxus BjFCN1 and human HmFCN2. The deduced protein of BjFCN1 is a canonical ficolin, which consists of an N-terminal signal peptide, a middle collagen (COL) region, and a C-terminal fibrinogen-like (FBG) domain. Homo sapiens, Hm; Mus musculus, Mm; Xenopus laevis, Xe; Ascidiacea, Halocynthia roretzi, As. The sequences used in the alignment are indicated as follows: HmFCN2 (NP_004099); HmFCN3 (NP_003656); MmFCN1 (NP_032021); XeFCN3 (NP_001079138); AsFCN1 (BAB60704); AsFCN3 (BAB60706).

FIGURE 2.

Tissue distributions of amphioxus BjFCN1 (D–F), BjMASP1 (G–I), BjMASP3 (J–L), and BjC3 (M–O) detected by section in situ hybridization. The negative control is showed in A–C. Endostyle, e; gill, g; hepatic cecum, hc; intestine, i; muscle, m; notochord, nc; skin, s; spinal cord, sc; testis, t; ovary, o. The bar indicates 200 μm.

FIGURE 3.

Expression profiles of BjFCN1 (A), BjC3 (B), and BjMASP1/3(C) detected by qRT-PCR after challenge with purified pathogens LPS and LTA, bacteria S. aureus and V. anguillarum. Endogenous control for qualification was cytoplasm GAPDH.

FIGURE 4.

Purification and characterization of recombinant BjFCN1 protein. A, Flag-tagged recombinant BjFCN1 protein was subjected to non-reducing SDS-PAGE (12%) (left panel) and immunoblotted with anti-Flag mAb (right panel). BjFCN1 formed monomer (1mer), dimer (2mer), and tetramer (4mer) with disulfide bonds based on our analysis. B, His-tagged recombinant BjFCN1 protein was subjected to SDS-PAGE (12%) (left panel) and immunoblotted with anti-His mAb (right panel). C, hemagglutinin activity analysis of His-tagged recombinant BjFCN1 protein. TRX as a negative control was tested in parallel. D, binding of His-tagged recombinant BjFCN1 protein to GlcNAc and lactose agarose. Rough BjFCN1 protein (30 μg) alone as control was also subjected to SDS-PAGE.

FIGURE 5.

The interaction of His-tagged recombinant BjFCN1 protein and bacteria. A, micro-organism binding analysis of BjFCN1 protein. BjFCN1 bound to A. calcoaceticus (a), E. coli (b), K. pneumoniae (c), B. subtilis (d), V. anguillarum (e), S. aureus (f), S. hemeolyticus (g), S. saprophyticus (h), and E. faecalis (i) with calcium, while 10 mm EDTA completely inhibited the activation. BjFCN1 protein (15 μg) was a positive control (j). B, microbial aggregation analysis of BjFCN1 protein. BjFCN1 strongly aggregated S. aureus and weakly aggregated B. subtilis, S. hemeolyticus, and E. faecalis with the presence of calcium, while the effect was inhibited with EDTA. The bar on the micrographs indicates 100 μm. TRX protein (27 kDa) as a negative control was tested in parallel.

FIGURE 6.

A, architecture of amphioxus BjMASP1 and BjMASP3, human HmMASP1 and HmMASP3. B, GST-tagged recombinant BjMASP1/3-N protein was subjected to SDS-PAGE (12%) (left panel) and immunoblotted with anti-GST mAb (right panel). C, binding of His-tagged recombinant BjFCN1 protein to GST-tagged recombinant BjMASP1/3-N protein. BjFCN1 protein formed a complex with BjMASP1/3-N. D, His-tagged recombinant BjMASP1-C protein was subjected to SDS-PAGE (12%) (left panel) and immunoblotted with anti-His mAb (right panel).

FIGURE 7.

A, architecture of amphioxus BjC3 and human HmC3. α2M, α-2-macroglobulin domain; ANA, anaphylatoxin homologous domain; TE, thiol-ester_cl domain; C345C, netrin C-terminal domain. B, humoral BjC3 cleavage analysis. The protein mixtures described were analyzed by Native PAGE (left panels) and SDS-PAGE (right panels) under non-reducing conditions, and anti-BjC3 mAb was used in Western blotting. The presence of BjMASP1-C protein altered the molecular size of humoral BjC3 under both calcium and EDTA conditions, suggesting that BjMASP1-C protein exhibited proteolytic function on humoral BjC3. C, bacterial binding analysis of humoral BjC3 by Western blotting under reducing condition. Humoral fluid alone as a positive control was tested (a and k). BjC3 bound to A. calcoaceticus (b), K. pneumoniae (d), B. subtilis (e), V. anguillarum (f), S. aureus (g), S. hemeolyticus (h), and E. faecalis (j) under calcium condition, and 10 mm EDTA completely inhibited the activation.

FIGURE 8.

The comparison of amphioxus complement lectin pathway with human complement lectin pathway. The complement lectin-mediated pathway of amphioxus consists of FCN-MASP complex, C3, CCP models, and C6-like molecule.