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. 2016 Dec 23:6:39287.
doi: 10.1038/srep39287.

CsMAP34, a teleost MAP with dual role: A promoter of MASP-assisted complement activation and a regulator of immune cell activity

Mo-Fei Li  1   2 , Jun Li  3 , Li Sun  1   2
Affiliations

CsMAP34, a teleost MAP with dual role: A promoter of MASP-assisted complement activation and a regulator of immune cell activity

Mo-Fei Li et al. Sci Rep. .

Abstract

In teleost fish, the immune functions of mannan-binding lectin (MBL) associated protein (MAP) and MBL associated serine protease (MASP) are scarcely investigated. In the present study, we examined the biological properties both MAP (CsMAP34) and MASP (CsMASP1) molecules from tongue sole (Cynoglossus semilaevis). We found that CsMAP34 and CsMASP1 expressions occurred in nine different tissues and were upregulated by bacterial challenge. CsMAP34 protein was detected in blood, especially during bacterial infection. Recombinant CsMAP34 (rCsMAP34) bound C. semilaevis MBL (rCsBML) when the latter was activated by bacteria, while recombinant CsMASP1 (rCsMASP1) bound activated rCsBML only in the presence of rCsMAP34. rCsMAP34 stimulated the hemolytic and bactericidal activities of serum complement, whereas anti-CsMAP34 antibody blocked complement activities. Knockdown of CsMASP1 in C. semilaevis resulted in significant inhibition of complement activities. Furthermore, rCsMAP34 interacted directly with peripheral blood leukocytes (PBL) and enhanced the respiratory burst, acid phosphatase activity, chemotactic activity, and gene expression of PBL. These results indicate for the first time that a teleost MAP acts one hand as a regulator that promotes the lectin pathway of complement activation via its ability to recruit MBL to MASP, and other hand as a modulator of immune cell activity.

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Figures

Figure 1
Figure 1. Alignment of the sequences of CsMAP34 (A) and CsMASP1 (B) homologues.
Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsMAP34/CsMASP1 and the compared sequences. The consensus residues are in blue, the residues that are ≥75% identical among the aligned sequences are in grey. Red, yellow, blue, and green boxes indicate CUB domain, calcium-binding EGF-like domain, complement control protein (CCP) module, and trypsin-like serine protease domain, respectively. The GenBank accession numbers of the aligned sequences are as follows: (A) Maylandia zebra (zebra mbuna), XP_004554228.1; Haplochromis burtoni (African cichlid fish), XP_005915044.1; Larimichthys crocea (large yellow croaker), XP_010729754.1; Stegastes partitus (bicolor damselfish), XP_008280824.1; Poecilia Mexicana (Atlantic molly), XP_014846064.1; Cyprinodon variegates (sheepshead minnow), XP_015233610.1; Poecilia reticulate (guppy), XP_008411647.1; Notothenia coriiceps (Antarctic bullhead notothen), XP_010779504.1; Salmo salar (Atlantic salmon), XP_014001301.1; Oncorhynchus mykiss (rainbow trout), CDQ81624.1; Esox lucius (northern pike), XP_010890090.1. (B) Haplochromis burtoni (African cichlid fish), XP_014184888.1; Oreochromis niloticus (Nile tilapia), XP_005462796.1; Maylandia zebra (zebra mbuna), XP_014266317.1; Larimichthys crocea (large yellow croaker), XP_010748082.1; Austrofundulus limnaeus (annual killifish), XP_013886517.1; Fundulus heteroclitus (Atlantic killfish), XP_012712429.1; Poecilia latipinna (Poecilia latipinna), XP_014894567.1; Poecilia Mexicana (Atlantic molly), XP_014836495.1; Salmo salar (Atlantic salmon), XP_014068886.1; Esox lucius (northern pike), XP_012988193.1; Oncorhynchus mykiss (rainbow trout), NP_001153950.1; Astyanax mexicanus (blind cavefish), XP_007233578.1; Cyprinus carpio (common carp), BAA86866.1.
Figure 2
Figure 2. Expression of CsMAP34 and CsMASP1 in fish tissues under different conditions.
(A) Under normal conditions, CsMAP34 (Aa) and CsMASP1 (Ab) expression in the intestine, gill, heart, spleen, brain, muscle, liver, blood, and kidney of tongues sole was determined by quantitative real time RT-PCR (qRT-PCR). For convenience of comparison, the expression levels in intestine were set as 1. (B) C. semilaevis were infected with or without (control) Vibrio anguillarum, and CsMAP34 (Ba) and CsMASP1 (Bb) expression in blood was determined by qRT-PCR at various time points. In all cases, data are the means of three independent experiments and presented as means ± SEM. *P < 0.05, **P < 0.01.
Figure 3
Figure 3. Binding of rCsMAP34 and rCsMASP1 to MBLs (A) and bacteria (B).
(A) rCsMAP34, rCsMASP1, and rTrx were incubated with rCsBML1, rCsBML2, or rCsBML3 in the presence of Vibrio anguillarum and calcium. For control, rCsMAP34, rCsMASP1, and rTrx were incubated with HBSS in the presence of Vibrio anguillarum and calcium. Protein-protein binding was determined by ELISA. Data are the means of three independent experiments and presented as means ± SEM. **P < 0.01. (B) rCsMAP34 was incubated with V. anguillarum alone (Bg and Bh) or with V. anguillarum plus rCsBML3 in the presence (Ba and Bb) or absence (Bd and Be) of calcium, and bacteria-associated rCsMAP34 was detected by FITC-labeled antibody and stained with DAPI. Bc, a merged image of Ba and Bb; Bf, a merged image of Bd and Be; Bi, a merged image of Bg and Bh.
Figure 4
Figure 4. Effect of rCsMAP34 antibody and rCsMAP34 on complement activation.
(A and B) Cynoglossus semilaevis serum in various dilutions was incubated with or without (control) rCsMAP34 antibody, rTrx antibody, or preimmune antibody. At 1 h after incubation, the hemolytic (A) and bactericidal (B) activities of the serum were determined. (C and D) C. semilaevis serum in various dilutions was incubated with or without (control) rCsMAP34 plus activated rCsBML3, rCsMAP34 plus rTrx, rCsMAP34 alone, activated rCsBML3 plus rTrx, activated rCsBML3 alone, or rTrx. At 1 h after incubation, the hemolytic (C) and bactericidal (D) activities of the serum were determined. Data are the means of three independent assays and presented as means ± SEM. *P < 0.05; **P < 0.01.
Figure 5
Figure 5. Effect of CsMASP1 knockdown on complement activation.
Serum from Cynoglossus semilaevis treated with pCsMASP1si, pCsMASP1siC, or PBS (control) was serially diluted and determined for hemolytic activity. Data are the means of three independent assays and presented as means ± SEM. **P < 0.01.
Figure 6
Figure 6. Activation of peripheral blood leukocytes (PBL) by rCsMAP34.
(A) Cynoglossus semilaevis PBL were incubated with or without (control) rCsMAP34 or rTrx for 1 h, and immune gene expression was determined by quantitative real time RT-PCR. (B and C) C. semilaevis PBL were incubated as above for various hours, and the cells were then determined for respiratory burst (B) and acid phosphatase activity (C). (D) The chemotactic activity of rCsMAP34 or rTrx in various concentrations against PBL was determined using transwell migration assay. Data are the means of three independent assays and presented as means ± SEM. *P < 0.05; **P < 0.01.
Figure 7
Figure 7. A proposed functional model of CsMAP34 and CsMASP1 in the lectin pathway activation of Cynoglossus semilaevis.
Following pathogen infection, BML recognizes and binds the pathogen; CsMAP34 interacts with the pathogen-bound MBL and recruits CsMASP1; association with the CsMAP34-MBL complex activates CsMASP1 and leads to further activation of the lectin pathway cascade.
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