MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

Abstract

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

Figure 1. Development of the MASP-3 specific inhibitor, TFMI-3.

(A) The TFPI-1 domain 2 (TFPI-D2) sequence around the P1 (135) position. The numbering corresponds to the full length protein sequence from the UniProt database. (B) Codon-bias normalized sequence logo of MASP-3-selected unique clones from the TFPI-D2 phage library. Position heights represent the degree of conservation. The nonrandomized Cys residue shows the maximum height. Letter heights indicate normalized amino acid frequencies. Colors reflect chemical properties.

Figure 2. Interaction of TFMI-3 and MASP-3 determined by Surface Plasmon Resonance.

MASP-3cf was injected over TFMI-3_HA immobilized on anti-HA antibody-coated GLM sensor chip as described in “Experimental Procedures”. Result of one of the three independent runs is shown as sensograms corresponding to five parallel analyte injections of different MASP-3cf concentrations. Smooth lines represent global fit of the experimental data to a 1:1 Langmuir model.

Figure 3. Lack of inhibitory effect of TFMI-3 on the three complement pathways.

TFMI-3 was applied in 0–3.5 μM in standard complement activation assays (Wieslab). No significant inhibition of any of the three pathways, CP (■), LP (●) or AP (▲) was observed in normal human serum. Typical curves from 3 parallel assays are shown where each data point represents the mean of 3 repeats. SD bars are not shown to allow distinction of the symbols.

Figure 4. Inhibition of pro-FD activation by TFMI-3.

Conversion of Cy3-labeled pro-FD to Cy3-FD (referred to as activation) in different types of normal human blood preparations was detected by reducing SDS-PAGE followed by fluorimetric scanning and densitometry. Representative gels out of at least 2 parallels are shown on the left side. On the right side of each panel densitometric analysis of the pro-FD and the FD bands of the same gel is shown. The intersection point of the dashed trend lines gives the half-life of pro-FD. Calculated half-lives are found in s 3. (A) Activation of Cy3-pro-FD in citrated normal human plasma. (B) Lack of significant activation of Cy3-pro-FD in citrated normal human plasma in the presence of 1000 nM TFMI-3. (C) Activation of Cy3-pro-FD in normal human EDTA plasma. (D) Lack of significant activation of Cy3-pro-FD in normal human EDTA plasma in the presence of 1000 nM TFMI-3. (E) Activation of Cy3-pro-FD in hirudin-treated normal human plasma. (F) Lack of significant activation of Cy3-pro-FD in normal human hirudin plasma in the presence of 1000 nM TFMI-3. (G) Activation of Cy3-pro-FD in normal human serum. (H) Delayed activation of Cy3-labelled pro-FD in normal human serum in the presence of 1000 nM TFMI-3.