A novel mannose-binding lectin/ficolin-associated protein is highly expressed in heart and skeletal muscle tissues and inhibits complement activation

Abstract

The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1 isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of quantitative PCR and MAP-1-specific immunohistochemistry, we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues as well as in liver hepatocytes with a different expression profile than that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, ficolin-2, and ficolin-3, and recombinant MAP-1 was able to inhibit complement C4 deposition via both the ficolin-3 and MBL pathway. In conclusion we have identified a novel 45-kDa serum protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and ficolins and may function as a potent inhibitor of the complement system in vivo.

FIGURE 1.

Alignment of the isoforms of MASP1. The deduced amino acid sequences of MASP-1 (MASP1 isoform 1), MASP-3 (MASP1 isoform 2), and MAP-1 (MASP1 isoform 3) were aligned using the BioEdit Software. The dots represent identical amino acids. MASP-1 and MASP-3 contain different C-terminal serine protease domains, whereas MAP-1 does not contain a serine protease domain. Instead the protein contains 17 unique amino acids at the C-terminal end.

FIGURE 2.

Alternative splicing of the MASP1 gene. MASP1_v1 is generated by splicing out exons 9 and 12, both of which contain a stop codon sequence (marked with black boxes). The MASP1_v1 sequence contains a stop codon in exon 18. The MASP1_v2 is generated by splicing out exon 9 and terminated by the stop codon in exon 12. The MASP1_v3 is generated if exon 9 is not spliced out. The MAP-1 protein contains the exons encoding the two CUB domains, the EGF domain, and the first CCP domain together with the unique exon 9 domain.

FIGURE 3.

Quantitative tissue distribution of MASP1_v1, MASP1_v2, and MASP1_v3. The tissue distributions of the MASP1_v1, MASP1_v2, and MASP1_v3 transcripts were investigated using real time qPCR. A very high expression of MASP1_v3 was detected in the heart and skeletal muscle tissues. The MASP1_v3 expression was higher in liver compared with MASP1_v1 and MASP1_v2. The transcription was normalized to the mean of BCR, GAPDH, and β-actin housekeeping genes. The relative expression of the splice variants of MASP1 was calculated with the MASP1_v3 expression in the heart as reference (index, 100). The data were corrected for differences in amplification efficiencies. The qPCR system was performed in duplicate.

FIGURE 4.

Antibody specificities. A, rMASP-1, rMASP-3, and rMAP-1 (MASP1 isoforms 1, 2, and 3, respectively) were immobilized in 2-fold serial dilutions on Maxisorp plates at a starting concentration of 5 μg/ml. Immunodetection was performed with anti-MAP-1 (mAb 20C4; left side) and with anti-MASP-1/3 (mAb 8B3; right side). Immunodetection values are given as A_{490–650 nm}. The error bars indicate two times the standard deviation of double determinations. B, SDS-PAGE/electroblotting of rMASP-1, rMASP-3, and rMAP-1. Immunodetection was performed with anti-MAP-1 (mAb 20C4; left side) and anti-MASP-1/3 (mAb 8B3; right side). C, immunohistochemical analysis of formaldehyde fixed CHO cells expressing rMASP-3 or rMAP-1. Immunodetection was performed with anti-MAP-1 (mAb 12B11; left and middle section) and with anti-MASP-1/3 (mAb 8B3; right section).

FIGURE 5.

Immunohistochemical analysis of MAP-1 (MASP1 isoform 3) tissue localization. The left panels show staining with the specific mAb 12B11 to MAP-1. The right panels show the isotype control staining with a nonrelated IgG1k mAb. A and B, myocardium; C and D, skeletal muscle; E and F, liver tissue; G and H, aortic tissue. The bottom right corner bar indicates 50 μm on all slides.

FIGURE 6.

Immunoprecipitation of MAP-1 (MASP1 isoform 3) and MASP-1/3 (MASP-1 isoform 1 and 2) serum complexes. A, MAP-1 and MASP-1 and -3 were immunoprecipitated from serum using anti-MAP-1 (mAb 20C4) and anti-MASP-1/3 (mAb 8B3). Serum immunoprecipitation with an IgG1κ isotype antibody served as a negative control. Reduced samples were applied to SDS-PAGE, and immunoblotting was detected with biotinylated mAbs to ficolin-3 (FCN313), MBL (Hyb 131-11), or MAP-1 (mAb 20C4). B, immunoprecipitation with mAbs to MBL (Hyb 131-11), ficolin-2 (FCN219), and ficolin-3 (FCN334) from 1 ml, 300 μl, and 100 μl of serum, respectively (left side). Positive controls were MAP-1 precipitated from 100 μl serum and rMAP-1 from 1 ml of culture supernatant using mAb 20C4 (right side). Immunoprecipitation from 1 ml of serum with an IgG1κ isotype antibody served as negative control. The samples were analyzed by immunoblotting probed with a biotinylated antibody to MAP-1.

FIGURE 7.

Influence of rMASP-2 (MASP2 isoform 1) and rMAP-1 (MASP1 isoform 3) on the MBL- and ficolin-3-mediated complement C4 deposition. Polystyrene plates were coated with either mannan followed by 400 ng/ml rMBL or AcBSA followed by 400 ng/ml rficolin-3. MBL- or ficolin-3-deficient serum was added in dilutions from 0.01 to 2.5%. C4 depositions were measured using a polyclonal antibody to C4 and are given as A_{490–650 nm} values. The error bars indicate two times the standard deviation of double determinations. Approximated concentrations of rMBL, rficolin-3, rMAP-1, and rMASP-2 are given in the figure labels. A–C show MBL-dependent C4 deposition on mannan, whereas D–F show ficolin-3-dependent C4 deposition on AcBSA. A, C4 deposition on a mannan-coated surface incubating rMBL at 400 ng/ml before addition of MBL-deficient serum in different dilutions. The control was without the addition of rMBL. B, dose-dependent enhancing effect of rMASP-2 on the rMBL-mediated C4 deposition. A total of 400 ng/ml of rMBL was preincubated with serial dilutions of rMASP-2 prior to the application of MBL-deficient serum as described under “Experimental Procedures.” C, dose-dependent inhibitory effect of rMAP-1 on the rMBL-mediated C4 deposition. A total of 400 ng/ml of rMBL was preincubated with serial dilutions of rMAP-1 prior to the application of MBL-deficient serum as described under “Experimental Procedures.” D, C4 deposition on an AcBSA-coated surface incubating rficolin-3 at 400 ng/ml before the addition of ficolin-3-deficient serum in different dilutions. The control was without addition of rficolin-3. E, dose-dependent enhancing effect of rMASP-2 on the rficolin-3-mediated C4 deposition. A total of 400 ng/ml of rficolin-3 was preincubated with serial dilutions of rMASP-2 prior to the application of ficolin-3-deficient serum as described under “Experimental Procedures.” F, dose-dependent inhibitory effect of rMAP-1 on the rficolin-3-mediated C4 deposition. A total of 400 ng/ml of rficolin-3 was preincubated with serial dilutions of rMAP-1 prior to the application of ficolin-3-deficient serum as described under “Experimental Procedures.”

FIGURE 8.

Influence of rMASP-2 (MASP2 isoform 1) and rMAP-1 (MASP1 isoform 3) on the complement C4 deposition in a pure system. A total of 400 ng/ml of rMBL was applied to a mannan surface followed by incubation for 45 min with serial dilutions of rMASP-2 in the first dimension. Afterward the serial dilutions of rMAP-1 were applied in the second dimension followed by application of purified C4 at 1 μg/ml. The C4 depositions (given as A_{490–650 nm} values) were measured with a polyclonal antibody to C4. The error bars indicate two times the standard deviation of double determinations. Approximated concentrations of rMAP-1 and rMASP-2 are given in the figure labels.