Visual Abstract

Factor D (FD) is an essential element of the alternative pathway of the complement system, and it circulates predominantly in cleaved, activated form in the blood. In resting blood, mannose-binding lectin–associated serine protease 3 (MASP-3) is the exclusive activator of pro-FD. Similarly to FD, MASP-3 also circulates mainly in the active form. It was not clear, however, how zymogen MASP-3 is activated. To decipher its activation mechanism, we followed the cleavage of MASP-3 in human hirudin plasma. Our data suggest that neither lectin pathway proteases nor any protease controlled by C1-inhibitor are required for MASP-3 activation. However, EDTA and the general proprotein convertase inhibitor decanoyl-RVKR-chloromethylketone completely prevented activation of exogenous MASP-3 added to blood samples. In this study, we show that proprotein convertase subtilisin/kexin (PCSK) 5 and PCSK6 are able to activate MASP-3 in vitro. Unlike PCSK5, PCSK6 was detected in human serum and plasma, and previously PCSK6 had also been shown to activate corin in the circulation. In all, PCSK6 emerges as the MASP-3 activator in human blood. These findings clarify the very first step of the activation of the alternative pathway and also connect the complement and the proprotein convertase systems in the blood.

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The complement system is an essential arm of the innate immune repertoire. It is activated by three interconnected routes: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). LP components are large Ca2+-dependent macromolecular complexes with varying composition (13). A typical complex is composed of a pattern recognition molecule (PRM) and a Ca2+-dependent serine protease dimer. PRMs of the LP include mannose-binding lectin (MBL); ficolins 1, 2, and 3 (also known as [aka] M-, L-, H-ficolins); collectin kidney 1 (CL-K1, aka CL-11); and collectin liver 1 (CL-L1, aka CL-10). Beside the three MBL-associated serine proteases (MASP-1, MASP-2, and MASP-3), two regulatory, nonenzymatic MBL-associated proteins (MAp19 and MAp44) complete the list of LP proteins in humans. When the PRM components bind to activating carbohydrate patterns, first MASP-1 autoactivates, then it activates MASP-2 (46), and the activation progresses via the common CP/LP route, leading to the formation of the CP/LP C3-convertase, C4b2a. The AP, which might be initiated on its own (7), serves as an amplificatory loop regardless of the initial activation route. The serine protease, factor D (FD), is an essential component of the AP. FD cleaves C3b-bound factor B (FB) to produce the labile AP C3-convertase, C3bBb, which will generate further C3b molecules (8, 9).

Until recently, FD had been thought to be activated at the site of its synthesis, and the function of MASP-3 had been merely regarded as a negative regulator (1012). By now, it has become clear that FD is produced in the zymogen (pro-FD) form, and MASP-3 functions as the professional pro-FD activator in humans in the circulation (1315). A fresh report provided in vivo evidence that this function of MASP-3 is evolutionarily conserved in humans and mice (16). Only the active form of MASP-3 can cleave pro-FD, and we have shown earlier that MASP-3 circulates primarily as an active enzyme in human blood (17, 18); nonetheless, its activation mechanism remained unknown.

It is intriguing that MASP-3, which circulates mostly in the active form, cannot autoactivate, whereas MASP-1, which has a profound autoactivation capacity, is proenzymic in the blood in the absence of LP activators. Notably, MASP-1 and MASP-2 are regulated by serpins, primarily by C1-inhibitor, whereas MASP-3 has no known physiological inhibitor. In vitro, both MASP-1 and MASP-2 can activate MASP-3, and even the proenzyme form of MASP-1 is able to do so with a low efficiency. Earlier we outlined a theoretical activation mechanism within the framework of the LP; however, as an alternative hypothesis, we proposed activation of MASP-3 by paired basic amino acid–specific proprotein convertases (PBA-PCs), possibly intracellularly (18).

In humans, proprotein convertases are encoded by a total of nine genes. Most of them act intracellularly and belong to the subtilisin/kexin family of serine proteases. They are Ca2+-dependent enzymes, and seven of the nine members cleave preferably after paired basic residues, with the consensus sequence being R/K-X0,2,4,6-R/K (19). A coherent nomenclature exists for this group of enzymes. In humans they are called proprotein convertase subtilisin/kexin type 1–9 (PCSK1–9). Less-consistent earlier names are also in use. Although the primary role of proprotein convertases is the intracellular processing of various hormones and proteins, some of them might be present and act extracellularly, including in the bloodstream. Furin (PCSK3), the prototype mammalian enzyme, is a membrane protein that recycles from the trans-Golgi to the cell surface and back; however, some of it might get into the bloodstream by shedding (20). PCSK6 (aka paired basic amino acid–cleaving enzyme 4 [PACE4]), which is secreted into the extracellular space (21), has been shown to activate corin, a regulator of blood pressure through the production of natriuretic peptides, in the blood (22, 23). PCSK5 has two isoforms. One of them (termed PC5B/PC6B) is a membrane-bound enzyme; however, the other (PC5A/PC6A) is a secreted enzyme (21). Finally, PCSK7 is a ubiquitous member of the family (21), which has a similar structure to furin, and some of it might also get into the bloodstream by shedding. In summary, four of the seven PBA-PCs can be potentially present in the blood.

In this study we aimed to decipher the activation mechanism of human MASP-3. We found that MASP-3 is efficiently activated in the blood, the activation is not autoactivation, and neither LP proteases nor any protease controlled by C1-inhibitor nor PRM binding are required for MASP-3 activation. Our data point toward a mechanism involving one or more PBA-PCs that are present in the blood.

Benzamidine, Pefabloc SC, PMSF, p-nitrophenyl-p′-guanidinobenzoate (NPGB), and 1,10-phenanthroline were from Merck/Sigma-Aldrich. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and anti–MASP-3 Ab (MAB1724) were from R&D Systems. CNBr-activated Sepharose 4 Fast Flow and Cy5 monoreactive NHS-ester were from GE Healthcare.

Active recombinant MASP-1 catalytic fragment (cf), zymogen and active MASP-3cf, MASP-1_D1-3 (Fig. 1), and active human FD were produced as described (5, 17, 2427). SGMI-1 and SGMI-2 were prepared according to Héja et al. (28). Recombinant human MBL (rMBL), produced based on the protocol of Vorup-Jensen et al. (29), was from Enzon Pharmaceuticals. MBL-Sepharose (1.6–2.5 mg of rMBL per milliliter of wet resin) was prepared as described (18).

FIGURE 1.

Domain structure of MASPs and the recombinant fragments used in this study. Full-length MASPs are composed of six domains. Five regulatory domains (C1r/C1s-Uegf-BMP [CUB] domain; epidermal growth factor [EGF] domain; complement control protein [CCP] domain) are followed by a serine protease (SP) domain. Activation requires cleavage at a specific Arg-Ile bond. The two chains are held together by a disulfide bond. Under reducing conditions the two chains run separately. Under nonreducing conditions they remain together; however, the mobility of the active form is slightly different from that of the zymogen form when analyzed by SDS-PAGE.

FIGURE 1.

Domain structure of MASPs and the recombinant fragments used in this study. Full-length MASPs are composed of six domains. Five regulatory domains (C1r/C1s-Uegf-BMP [CUB] domain; epidermal growth factor [EGF] domain; complement control protein [CCP] domain) are followed by a serine protease (SP) domain. Activation requires cleavage at a specific Arg-Ile bond. The two chains are held together by a disulfide bond. Under reducing conditions the two chains run separately. Under nonreducing conditions they remain together; however, the mobility of the active form is slightly different from that of the zymogen form when analyzed by SDS-PAGE.

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The full-length MASP-3 gene was amplified from pEAK8–MASP-3 (12) by PCR, and the product was cloned into pVL1392 (BD Biosciences) at the BglII and EcoRI sites. The S664A (precursor numbering) mutant (MASP-3–S/A) was made by mutagenesis (QuikChange Lightning Multi kit, Agilent).

The same protocol was used for the purification of both wild-type (zymogenic) MASP-3 and MASP-3–S/A. Sf9 cells were cotransfected with pVL1392-MASP3 applying the flashBAC GOLD protocol (Oxford Expression Technologies). Viruses were amplified, then recombinant MASP-3 was expressed in ∼2 × 109Sf9 cells using a multiplicity of infection of ∼2. After 3 d of shaking at 27°C, cells were removed (500 × g, 5 min), and PMSF (1 mM final) was added to the filtered supernatant.

The supernatant was diluted 2-fold with 50 mM Tris, 150 mM NaCl, 1 mM PMSF, pH 8.0, applied to a 16 × 100-mm Q-Sepharose High Performance (GE) column, and eluted with a 150- to 500-mM NaCl gradient in 50 mM Tris and 5 mM CaCl2, pH 8.0. MASP-3–containing fractions were applied to a 5 × 93-mm MBL-Sepharose column equilibrated with 50 mM Tris, 500 mM NaCl, and 5 mM CaCl2, pH 8.0. The column was washed with 50 mM Tris and 150 mM NaCl, pH 8.0, then the bound proteins were eluted with a combined 150–650 mM NaCl, 0–5 mM EDTA linear gradient in 50 mM Tris, pH 8.0. Pure MASP-3–containing fractions were diluted 2-fold with 50 mM Tris and 20 mM CaCl2, pH 8.0, dialyzed overnight against HBS-Ca (50 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4) at 4°C, concentrated to ∼250 μg/ml, and stored frozen in aliquots. MASP-3 concentration was calculated using the ε280 = 118,285 M–1cm–1 and 79.6 kDa (not accounting for glycosylation) values.

Labeling of MASP-3 variants was performed in their storage buffers: HBS-Ca for MASP-3–S/A or HBS-EDTA (50 mM HEPES, 140 mM NaCl, 0.1 mM EDTA, pH 7.4) for MASP-3cf. The Cy5-NHS-ester to protein molar ratio was 2:1 (17). MASP-3–S/A–Cy5 (incorporated dye-to-protein ratio of ∼1.1) was purified on MBL-Sepharose, dialyzed in HBS-Ca, concentrated to 225 μg/ml, and stored frozen in aliquots. MASP-3cf–Cy5 (incorporated dye-to-protein ratio of ∼0.8) was purified on a 10 × 300-mm Superose 12 (GE) column in HBS-EDTA, concentrated to ∼180 μg/ml, and stored frozen in aliquots. MASP-3cf has an extinction coefficient of ε280 = 81,205 M–1 cm−1 and a molecular mass of 48.1 kDa. Labeled protein concentrations and labeling efficiencies were calculated as described (30).

MASP-3–S/A or MASP-3–S/A–Cy5 (both ∼2.5 μM) were incubated at 37°C in HBS-Ca with ∼500 nM MASP-1cf. MASP-3cf or MASP-3cf–Cy5 (both ∼3.6 μM) were incubated at 37°C in HBS-EDTA with ∼450 nM MASP-1cf. Samples were withdrawn at 0, 0.5, 1, 2, and 3 h. Reactions were stopped by SDS-PAGE sample buffer and heating (2 min, 95°C). Samples were analyzed by SDS-PAGE under reducing conditions.

MASP-3–S/A or MASP-3cf (both ∼1 μM) were incubated at 37°C with ∼1 μM active human FD in HBS-Ca or HBS-EDTA, respectively. Samples were withdrawn at 0, 0.25, 1, and 4 h. Reactions were stopped as above, then samples were analyzed by reducing SDS-PAGE.

Serum or plasma from 10 healthy donors were collected by vein puncture into S-Monovette (Sarstedt) tubes prepared with clot activator, K3-EDTA, or recombinant hirudin. Samples of the same type were combined, aliquoted, and stored at −80°C. Each aliquot was thawed only once. K3-EDTA plasma was individually collected from six type I HAE patients. The study was conducted in conformity with the World Medical Association Declaration of Helsinki, also approved by the local ethics committee (permission number: TUKEB 9190-1/2017/EKU). Informed consent was obtained from the donors.

MASP-3–S/A–Cy5 (final concentration of 5 μg/ml ∼60 nM) was added to hirudin or EDTA plasma, and the mixtures were incubated at 37°C for 24 h. Aliquots were withdrawn at every 1–2 h during the first 8–10 h, then at 24 h. Similar MASP-3–S/A–Cy5 cleavage assays were performed in hirudin plasma in the presence of the following additives (final concentrations and typical sampling times are indicated): MASP-1_D1-3, 320 μg/ml (10 μM), 0, 4, and 24 h; SGMI-1, 10 μM, 0, 4, and 24 h; SGMI-2, 10 μM, 0, 4, and 24 h; NPGB, 100 μM, 0, 4, and 24 h; benzamidine, 1 mM, 0, 4, and 24 h; Pefabloc, 10 mM, 0, 4, and 24 h; 1,10-phenanthroline, 10 mM, 0, 4, and 24 h; and dec-RVKR-cmk, 1 μM, every 1–2 h during the first 8 h and then 24 h. All additives were preincubated with hirudin plasma for 10–30 min at room temperature before the addition of MASP-3–S/A–Cy5.

Cleavage of MASP-3cf–Cy5 (∼5 μg/ml ∼100 nM final) was assayed similarly in hirudin plasma at 37°C. Samples were withdrawn at every hour during the first 8 h and then at 24 h.

All reactions were stopped by 10-fold dilution with SDS-PAGE sample buffer and heating (2 min, 95°C). MASP-3–S/A–Cy5–containing samples were analyzed under nonreducing conditions, whereas MASP-3cf–Cy5–containing samples were analyzed under reducing conditions by SDS-PAGE.

N-acetyl-d-glucosamine (D-GlcNAc)–agarose (Sigma-Aldrich, A2278), 100 μl, was washed with 1 ml of ice-cold HBS-Ca (see above). Hirudin plasma, 900 μl, was applied to the column in two cycles. The resin was washed with 1 ml of ice-cold HBS-Ca, and PRM-MASPs were eluted with 1 ml of ice-cold HBS-Ca supplemented with 200 mM D-GlcNAc. Finally, a wash with 1 ml of ice-cold 50 mM HEPES, 150 mM NaCl, and 20 mM EDTA, pH 7.4, was applied. Cleavage of MASP-3–S/A–Cy5 was assayed in the fractions as described above for plasma samples.

Enrichment of MASPs from EDTA plasma was performed as described (18) starting with 3 ml of individual plasma from HAE patients or pooled plasma from healthy volunteers. Activation of endogenous MASP-3 was quantified after nonreducing SDS-PAGE and Western blot (WB) (18).

MASP-3–S/A (final concentration 50 μg/ml) was added to hirudin plasma samples, and the mixtures were incubated at 37°C or 25°C. Aliquots were withdrawn at 0, 2, 4, 6, 8, 24, and 50 h. The same experiment was performed in EDTA plasma at 37°C for 24 h.

dec-RVKR-cmk (1 μM final) was added to hirudin plasma and preincubated for 10 min at room temperature, then MASP-3–S/A (50 μg/ml final) was added, and the mixture was incubated at 37°C for 24 h. Samples were withdrawn at 0, 2, 4, 6, and 8 h, then another aliquot of dec-RVKR-cmk (+1 μM final) was added, and a 24-h sample was collected.

All reactions were stopped by 10-fold dilution with SDS-PAGE sample buffer and heating (2 min, 95°C). Samples were analyzed by nonreducing SDS-PAGE and WB.

The genes encoding PCSK6 (isoform PACE4A-I) and PCSK5 (isoform PC5A/PC6A) were synthesized by Thermo Fisher Scientific. Both constructs were codon optimized and contained sequences encoding a short linker and a His-tag at the C terminus (Supplemental Table I). Genes were cloned into the pcDNA3.1(+) vector into the BamHI and NotI sites. The plasmid DNAs were used to transfect ExpiCHO cells using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific). Cells transfected with the empty vector were used as control. Purification of the recombinant PCSK enzymes was not successful; therefore, the CHO cell supernatants were used for the cleavage assays. Cells were maintained at 37°C and 8% CO2 in serum-free ExpiCHO expression medium for up to 4 d after transfection as suspension cultures. Maximal MASP-3–cleaving activity was detected at 1.5–2 d posttransfection, and then the activity deteriorated, possibly because of degradation of the enzymes at 37°C. Therefore, the cleavage assays were performed with cell supernatants harvested after 2 d. MASP-3cf–Cy5 (∼1 μg/ml ∼20 nM final) was added to the supernatants containing PCSK6 or PCSK5 and incubated at 25°C, and samples were withdrawn periodically as required for up to 48 h. Similar experiments were performed with the same supernatants containing PCSK6 or PCSK5 in the presence of 1 μM dec-RVKR-cmk and also with a supernatant of CHO cells transfected with the empty vector. Reactions were stopped by adding SDS-PAGE sample buffer and heating (2 min, 95°C), and then the samples were analyzed by reducing SDS-PAGE.

Table I.

The fraction of active MASP-3 in type I HAE patients and controls

SampleFraction of Active MASP-3 (%)
Healthy control (pooled plasma) 81 ± 4 
HAE patient no. 1 81 ± 6 
HAE patient no. 2 79 ± 7 
HAE patient no. 3 83 ± 3 
HAE patient no. 4 81 ± 5 
HAE patient no. 5 80 ± 5 
HAE patient no. 6 88 ± 2 
HAE patients average 82 ± 3a 
Healthy control (pooled plasma) 81 ± 4 
Healthy individuals average 81 ± 4b 
SampleFraction of Active MASP-3 (%)
Healthy control (pooled plasma) 81 ± 4 
HAE patient no. 1 81 ± 6 
HAE patient no. 2 79 ± 7 
HAE patient no. 3 83 ± 3 
HAE patient no. 4 81 ± 5 
HAE patient no. 5 80 ± 5 
HAE patient no. 6 88 ± 2 
HAE patients average 82 ± 3a 
Healthy control (pooled plasma) 81 ± 4 
Healthy individuals average 81 ± 4b 

Average ± SD from three parallels. The control pooled plasma value represents average from three independent isolations, whereas the individual patient samples were run three times using the same isolate.

a

Average and SD of the averages.

b

From Oroszlán et al. (‎18).

Fluorescent gels were scanned with a Typhoon laser scanner (GE Healthcare) or an Alliance Q9 mini (Uvitec) gel-doc system. Coomassie-stained gels were scanned in transparent mode, whereas WBs were scanned in reflective mode with an Epson Perfection 4490 scanner. Band intensities were quantified by densitometry using the Quantity One software (Bio-Rad).

The serum level of PCSK5 and the serum and hirudin plasma levels of PCSK6 were measured by sandwich ELISA using the kits MBS451559 and MBS9323189, respectively, from MyBioSource according to the protocols provided by the manufacturer.

To model MASP-3 activation in the blood we produced full-length (Fig. 1) recombinant human MASP-3 in the baculovirus-insect cell expression system. In agreement with a previous study (31), it was expressed as a one-chain proenzyme, and only minimal cleavage was observed. We replaced the active site serine to alanine (S664A), and most of the “activation” assays were performed with this inactive variant, which acts simply as a substrate. Purified MASP-3–S/A was labeled with Cy5 fluorescent dye. We used a reagent, which reacts with amino groups, at a concentration that allowed incorporation of ∼1 dye per protein molecule on average. The labeled protein enabled us to monitor the cleavage of low levels (∼5 μg/ml) of MASP-3, roughly equivalent to the physiological concentration (32). In vitro, MASP-3–S/A–Cy5 was efficiently cleaved by active MASP-1cf, indicating that labeling did not hinder cleavage by MASP-1 (Supplemental Fig. 1). Activation of MASP-3 gives rise to two bands corresponding to the “A” and “B” chains on reducing gels, whereas on nonreducing gels, the activated form runs slightly above the zymogen form (Supplemental Fig. 1C).

When added to hirudin plasma, MASP-3–S/A–Cy5 was cleaved by an enzyme with a half-life of 1–2 h (Fig. 2) at 37°C; however, the conversion was not complete, reaching a plateau of ∼30%. This could either mean that labeling interferes with the cleavage by the physiological activator or the activator is labile and inactivated at 37°C, or both. Cleavage is also efficient in serum (Supplemental Fig. 2A) but does not occur in EDTA plasma (see later).

FIGURE 2.

Cleavage of labeled recombinant MASP-3–S/A in hirudin plasma. Full-length recombinant S664A mutant MASP-3 labeled with Cy5 (MASP-3–S/A–Cy5) was added to human hirudin plasma at 5 μg/ml, and its cleavage at 37°C was followed by SDS-PAGE under nonreducing conditions. The cleaved (“activated”) form runs slightly above zymogen form. (A) Fluorescent scan of a typical gel. (B) Densitometric analysis of the same gel. Cleaved MASP-3–S/A–Cy5 ratio is defined as the percentage of density of the cleaved band compared with the combined density of the cleaved and zymogen bands.

FIGURE 2.

Cleavage of labeled recombinant MASP-3–S/A in hirudin plasma. Full-length recombinant S664A mutant MASP-3 labeled with Cy5 (MASP-3–S/A–Cy5) was added to human hirudin plasma at 5 μg/ml, and its cleavage at 37°C was followed by SDS-PAGE under nonreducing conditions. The cleaved (“activated”) form runs slightly above zymogen form. (A) Fluorescent scan of a typical gel. (B) Densitometric analysis of the same gel. Cleaved MASP-3–S/A–Cy5 ratio is defined as the percentage of density of the cleaved band compared with the combined density of the cleaved and zymogen bands.

Close modal

MASP-3 is a Ca2+-dependent dimer and forms Ca2+-dependent complexes with PRMs. Because EDTA (see later) blocked the activation of MASP-3–S/A–Cy5, we assumed that maybe only MASP-3 incorporated into complexes is activated in the blood. When we added the common interaction domains of MASP-1/3 (termed MASP-1_D1-3) in excess to hirudin plasma along with MASP-3–S/A–Cy5, we observed no difference compared with the control (Fig. 3A). We also purified PRM–MASP complexes on D-GlcNAc–agarose, which binds all PRMs (33, 34). After washing with Ca2+-containing buffer, elution was performed with D-GlcNAc in the presence of Ca2+, and then a final wash with EDTA was performed. We added MASP-3–S/A–Cy5 to the flow-through, to the wash, to the eluate, and the EDTA wash (Fig. 3B). Interestingly, the activator of MASP-3 was predominantly present in the flow-through and to a lesser extent in the wash fraction, and basically no activity was detected in the D-GlcNAc eluate containing the MASP–PRM complexes.

FIGURE 3.

Binding of MASP-3 to PRMs is not required for activation. (A) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma was followed as described in Fig. 2. Cleavage of MASP-3 also occurs in the presence of >100-fold molar excess of the D1-3 interaction domains of MASP-1/3 preventing binding of the labeled protein to PRMs. (B) Hirudin plasma was fractionated on D-GlcNAc–agarose. The D-GlcNAc eluate shall contain the PRM–MASP complexes. Cleavage of MASP-3–S/A–Cy5 was followed in the fractions similarly as described above. Most of the MASP-3–cleaving activity was detected in the flow-through and none in the eluate. (C) Activation of MASP-3cf–Cy5 (lacking the interaction domains) in hirudin plasma was followed by SDS-PAGE under reducing conditions. Fluorescent scan of a typical gel. (D) Densitometric analysis of the gel in (C). Cleaved MASP-3cf ratio is defined as the percentage of density of the cleaved bands (B chain + short chain) compared with the combined density of the cleaved and zymogen bands. The last data point was omitted from the fitting because of apparent degradation.

FIGURE 3.

Binding of MASP-3 to PRMs is not required for activation. (A) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma was followed as described in Fig. 2. Cleavage of MASP-3 also occurs in the presence of >100-fold molar excess of the D1-3 interaction domains of MASP-1/3 preventing binding of the labeled protein to PRMs. (B) Hirudin plasma was fractionated on D-GlcNAc–agarose. The D-GlcNAc eluate shall contain the PRM–MASP complexes. Cleavage of MASP-3–S/A–Cy5 was followed in the fractions similarly as described above. Most of the MASP-3–cleaving activity was detected in the flow-through and none in the eluate. (C) Activation of MASP-3cf–Cy5 (lacking the interaction domains) in hirudin plasma was followed by SDS-PAGE under reducing conditions. Fluorescent scan of a typical gel. (D) Densitometric analysis of the gel in (C). Cleaved MASP-3cf ratio is defined as the percentage of density of the cleaved bands (B chain + short chain) compared with the combined density of the cleaved and zymogen bands. The last data point was omitted from the fitting because of apparent degradation.

Close modal

We also labeled zymogen MASP-3cf, lacking the interaction domains, with Cy5, and followed its activation in hirudin plasma. Initial experiments indicated inefficient activation of labeled MASP-3cf; however, later it turned out to be caused by overlabeling. When we used MASP-3cf labeled with not more than one dye per protein molecule (on average), we observed activation kinetics (Fig. 3C, 3D), similar to the one obtained with the full-length protein.

These experiments suggest that binding to PRMs is not required for MASP-3 activation, labeling interferes with the cleavage, and the activator is probably not one of the MASPs.

Our initial hypothesis was that MASP-1 or MASP-2 may be responsible for MASP-3 activation. Because C1-inhibitor is the most important regulator of both MASP-1 and MASP-2, we assumed that C1-inhibitor deficiency could affect MASP-3 activation. C1-inhibitor has a broad specificity, it also inhibits kallikrein, fXIIa, fXIa, C1r, C1s, and plasmin (35). We enriched human plasma for MASPs on MBL-Sepharose and quantified MASP-3 activation by a WB-based method (18). We found that the ratio of active MASP-3 in samples from type I HAE patients was virtually identical to that from healthy individuals (Fig. 4A and Table I). These results are in accordance with those in the previous section, supporting that the MASP-3 activator is not MASP-1 or MASP-2. Moreover, they add that the MASP-3 activator is probably not an enzyme controlled by C1-inhibitor.

FIGURE 4.

MASP-1 and MASP-2 are not required for MASP-3 activation. (A) Detection of endogenous MASP-3 in enriched samples prepared from pooled plasma of healthy volunteers (control) or individuals with C1-inhibitor deficiency (no. 1–6). A typical WB is shown. Densitometric results are found in Table I. (B) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma was followed as described in Fig. 2. Cleavage of MASP-3–S/A–Cy5 also occurs in the presence of SGMI-1, an MASP-1–specific inhibitor, or SGMI-2, an MASP-2–selective inhibitor, both applied at a high concentration. Fluorescent scan of a typical gel.

FIGURE 4.

MASP-1 and MASP-2 are not required for MASP-3 activation. (A) Detection of endogenous MASP-3 in enriched samples prepared from pooled plasma of healthy volunteers (control) or individuals with C1-inhibitor deficiency (no. 1–6). A typical WB is shown. Densitometric results are found in Table I. (B) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma was followed as described in Fig. 2. Cleavage of MASP-3–S/A–Cy5 also occurs in the presence of SGMI-1, an MASP-1–specific inhibitor, or SGMI-2, an MASP-2–selective inhibitor, both applied at a high concentration. Fluorescent scan of a typical gel.

Close modal

The final proof that neither MASP-1 nor MASP-2 is the physiological MASP-3 activator came from experiments using our previously developed inhibitors (28). The activation of MASP-3–S/A–Cy5 in hirudin plasma was unaffected by the presence of SGMI-1, a MASP-1–specific inhibitor, or SGMI-2, a MASP-2–selective inhibitor (Fig. 4B). It is important to note that SGMI-1 was shown to inhibit not just the active form of MASP-1 but the low-level zymogen activity of MASP-1 as well (5), and SGMI-2 is also a weak inhibitor of MASP-3 (4). It also occurred to us that MASP-3 and FD may reciprocally activate each other; however, FD cleaved neither full-length MASP-3–S/A nor MASP-3cf (Supplemental Fig. 2D, 2E).

The above experiments indicate that the cleavage of MASP-3 is affected by covalent labeling because only ∼30% remained cleavable. Hence, we decided to follow the cleavage of unlabeled MASP-3. We found that we could easily detect the activation of exogenous recombinant MASP-3 added to plasma samples at supra-physiological concentration by WB using the same Ab as before (18).

At 37°C and 50 μg/ml, the half-life of uncleaved MASP-3–S/A was ∼4–8 h, and the cleaved fraction reached a plateau of ∼44% in 1 d in hirudin plasma (Fig. 5). This experiment indicated that the amount of the activating enzyme is probably limiting, and the activator lost its activity in 1 d at 37°C. At 25°C, the reaction was slower, but the activating enzyme remained active beyond 1 d. At day 2, the fraction of cleaved MASP-3–S/A was ∼53%, and the activation reaction had not plateaued yet (Fig. 5).

FIGURE 5.

Activation of unlabeled recombinant MASP-3. Unlabeled full-length recombinant S664A mutant MASP-3 (MASP-3–S/A) was added at high concentration (50 μg/ml) to human hirudin plasma, and its cleavage at 37°C or 25°C was followed by SDS-PAGE under nonreducing conditions. (A) A typical WB is shown. A faint band probably corresponding to endogenous active MASP-3 is also visible. Endogenous MASP-3 has a lower mobility compared with recombinant MASP-3 from insect cells because of different glycosylation. (B) Densitometric analysis of the same WB. Cleaved MASP-3–S/A ratio is defined as the percentage of density of the cleaved band compared with the combined density of the cleaved and zymogen bands. Activation of MASP-3 seems to have a lag phase at 25°C; therefore, the first two data points were omitted from the fitting. In contrast, the activator is more stable at 25°C than at 37°C.

FIGURE 5.

Activation of unlabeled recombinant MASP-3. Unlabeled full-length recombinant S664A mutant MASP-3 (MASP-3–S/A) was added at high concentration (50 μg/ml) to human hirudin plasma, and its cleavage at 37°C or 25°C was followed by SDS-PAGE under nonreducing conditions. (A) A typical WB is shown. A faint band probably corresponding to endogenous active MASP-3 is also visible. Endogenous MASP-3 has a lower mobility compared with recombinant MASP-3 from insect cells because of different glycosylation. (B) Densitometric analysis of the same WB. Cleaved MASP-3–S/A ratio is defined as the percentage of density of the cleaved band compared with the combined density of the cleaved and zymogen bands. Activation of MASP-3 seems to have a lag phase at 25°C; therefore, the first two data points were omitted from the fitting. In contrast, the activator is more stable at 25°C than at 37°C.

Close modal

In conclusion, the activating enzyme is heat labile and has a limiting concentration, and labeling indeed interferes with the activation because a higher fraction of unlabeled MASP-3 can be cleaved than that of the labeled one.

We observed already at the beginning that MASP-3 is not activated in EDTA plasma (Fig. 6). We also tested several general protease inhibitors: for example, the general metalloproteinase inhibitor 1,10-phenanthroline did not inhibit the reaction (Supplemental Fig. 2B), whereas NPGB, benzamidine, and Pefabloc partially inhibited the MASP-3 activator (Supplemental Fig. 2C). Initially we suspected that the Ca2+ dependence of MASP–PRM complexes is involved in the activation process, but this assumption has proved to be false (see before).

FIGURE 6.

Activation of recombinant MASP-3 is inhibited by EDTA or dec-RVKR-cmk. (A) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma or EDTA plasma was followed as described in Fig. 2. EDTA or dec-RVKR-cmk prevented “activation” of MASP-3–S/A–Cy5. Fluorescent scan of a typical gel. (B) Unlabeled MASP-3–S/A added to EDTA plasma or hirudin plasma supplemented with dec-RVKR-cmk was incubated and detected as described in Fig. 5. Again, “activation” of unlabeled MASP-3–S/A was completely blocked by EDTA or dec-RVKR-cmk. A typical WB is shown. See Fig. 5 as positive control.

FIGURE 6.

Activation of recombinant MASP-3 is inhibited by EDTA or dec-RVKR-cmk. (A) Cleavage of MASP-3–S/A–Cy5 in hirudin plasma or EDTA plasma was followed as described in Fig. 2. EDTA or dec-RVKR-cmk prevented “activation” of MASP-3–S/A–Cy5. Fluorescent scan of a typical gel. (B) Unlabeled MASP-3–S/A added to EDTA plasma or hirudin plasma supplemented with dec-RVKR-cmk was incubated and detected as described in Fig. 5. Again, “activation” of unlabeled MASP-3–S/A was completely blocked by EDTA or dec-RVKR-cmk. A typical WB is shown. See Fig. 5 as positive control.

Close modal

When we tested the general proprotein convertase inhibitor, dec-RVKR-cmk, in our MASP-3 activation assays, we found an explanation for our previous experiments. dec-RVKR-cmk completely prevented the activation of both labeled and unlabeled MASP-3–S/A in hirudin plasma (Fig. 6). Proprotein convertases are Ca2+-dependent and often heat-labile (36) serine proteases of the subtilisin/kexin fold and only poorly inhibited by general serine-protease inhibitors, like Pefabloc. Seven of the nine human proprotein convertases cleave after paired basic residues; hence, the MASP-3 activator must belong to this group. The activation site of MASP-3, (KR↓IIGGR) is quite similar to that of corin (KR↓ILGGR), an enzyme activated by PCSK6 (PACE4) in the blood. The lysine residue in the activation loop is probably easily accessible for labeling by Cy5-NHS-ester. This explains why only a fraction of labeled MASP-3 could be cleaved by the physiological activator. We found that PCSK6 is indeed present in human serum and hirudin plasma at ∼50–150 ng/ml (Supplemental Fig. 3). Very recently, others measured similar values (37). In contrast, we could not detect PCSK5 in human serum (data not shown).

PCSK6 (PACE-4) and PCSK5 (isoform PC5A aka PC6A) are the only secreted PBA-PCs. To test their ability to activate MASP-3, we expressed them in CHO cells. Both proteases were secreted but proved to be unstable to an extent that we could not purify them. Hence, we followed the activation of labeled MASP-3cf in the PCSK6- or the PCSK5-containing cell culture supernatants at 25°C (Figs. 7, 8). Both PCSK6 and PCSK5 activated MASP-3cf in this in vitro assay, as expected, but interestingly the PCSK5-containing supernatant was more efficient in this respect. The activation was blocked by the general PBA-PC inhibitor, dec-RVKR-cmk, in both cases, whereas only very low-level activation was observed in the supernatant of cells transfected with the empty vector.

FIGURE 7.

Cleavage of labeled MASP-3cf in supernatant of CHO cells expressing PCSK6. (A) Recombinant MASP-3cf–Cy5 was added at 1 μg/ml to supernatant of CHO expressing PCSK6, and the cleavage at 25°C was followed by SDS-PAGE under reducing conditions. Activation is indicated particularly by the appearance of the B chain of cleaved MASP-3cf–Cy5 (arrow) in the samples on the left side of the gel. Samples on the right side contained 1 μM dec-RVKR-cmk, which greatly reduced the cleavage to the background level. (B) As a control, MASP-3cf–Cy5 cleavage was monitored in CHO supernatant transfected with the empty vector. A very low-level background activation was observed, caused by an unknown CHO protease. Fluorescent scans of typical gels are shown.

FIGURE 7.

Cleavage of labeled MASP-3cf in supernatant of CHO cells expressing PCSK6. (A) Recombinant MASP-3cf–Cy5 was added at 1 μg/ml to supernatant of CHO expressing PCSK6, and the cleavage at 25°C was followed by SDS-PAGE under reducing conditions. Activation is indicated particularly by the appearance of the B chain of cleaved MASP-3cf–Cy5 (arrow) in the samples on the left side of the gel. Samples on the right side contained 1 μM dec-RVKR-cmk, which greatly reduced the cleavage to the background level. (B) As a control, MASP-3cf–Cy5 cleavage was monitored in CHO supernatant transfected with the empty vector. A very low-level background activation was observed, caused by an unknown CHO protease. Fluorescent scans of typical gels are shown.

Close modal
FIGURE 8.

Cleavage of labeled MASP-3cf in supernatant of CHO cells expressing PCSK5. Recombinant MASP-3cf–Cy5 was added at 1 μg/ml to supernatant of CHO expressing PCSK5, and the cleavage at 25°C was followed by SDS-PAGE under reducing conditions. Activation is indicated by the appearance of the two chains of cleaved MASP-3cf–Cy5 in the samples on the left side of the gel. Samples on the right side contained 1 μM dec-RVKR-cmk, which greatly reduced the cleavage to the background level. Fluorescent scan of a typical gel is shown.

FIGURE 8.

Cleavage of labeled MASP-3cf in supernatant of CHO cells expressing PCSK5. Recombinant MASP-3cf–Cy5 was added at 1 μg/ml to supernatant of CHO expressing PCSK5, and the cleavage at 25°C was followed by SDS-PAGE under reducing conditions. Activation is indicated by the appearance of the two chains of cleaved MASP-3cf–Cy5 in the samples on the left side of the gel. Samples on the right side contained 1 μM dec-RVKR-cmk, which greatly reduced the cleavage to the background level. Fluorescent scan of a typical gel is shown.

Close modal

Although the PCSK5-containing cell supernatant was more active in this in vitro assay, it might only reflect the different levels of expression by CHO cells and/or the different inherent stabilities of PCSK5 and PCSK6. What is important is that PCSK5 has not been detected in the blood. In contrast, PCSK6 has been shown by us and by others to be present in blood samples (37).

Overall, because of its MASP-3 activating capacity and in vivo localization, PCSK6 qualifies to be a physiological MASP-3 activator in the blood, whereas PCSK5 might serve as an MASP-3 activator at others sites of the body. Nevertheless, additional research is required to determine whether PCSK6 is the exclusive in vivo MASP-3 activator in the blood (Fig. 9.).

FIGURE 9.

The proposed activation mechanism of MASP-3. Our results support that MASP-3 is constitutively activated by a circulating proprotein convertase (or convertases) in the blood. The data are consistent with PCSK6 being the major activator in the blood; however, minor contribution of other enzymes having the same specificity cannot be completely excluded. Because active MASP-3 constitutively activates pro-FD of the alternative complement pathway, our discovery directly links proprotein convertases to the complement system as the highest-level activators of the AP in the blood. (Red arrows point from the enzyme to the substrate, whereas black arrows indicate conversion.)

FIGURE 9.

The proposed activation mechanism of MASP-3. Our results support that MASP-3 is constitutively activated by a circulating proprotein convertase (or convertases) in the blood. The data are consistent with PCSK6 being the major activator in the blood; however, minor contribution of other enzymes having the same specificity cannot be completely excluded. Because active MASP-3 constitutively activates pro-FD of the alternative complement pathway, our discovery directly links proprotein convertases to the complement system as the highest-level activators of the AP in the blood. (Red arrows point from the enzyme to the substrate, whereas black arrows indicate conversion.)

Close modal

MASP-3, which is traditionally regarded as a component of the LP, has been shown to be an essential constituent of the complement system, producing active FD, a key enzyme for the AP (1317). Only the active form of MASP-3 can carry out this cleavage (17), and MASP-3 was shown to circulate mostly as an active enzyme in humans and mice (18). However, a fraction of MASP-3 is proenzymic in the blood (∼20%); therefore, it is reasonable that MASP-3 is secreted into the blood as a zymogen, where it is activated via an unknown mechanism. Another possibility is that partial activation occurs intracellularly; however, this scenario is unlikely because recombinant MASP-3 has always been produced as a zymogen in eukaryotic cells (31, 38, 39). We and others have shown that MASP-3 lacks any capacity to autoactivate as an isolated enzyme (18, 31). MASP-3 has no known physiological inhibitor; it simply has a very narrow substrate specificity, which allows MASP-3 to be present as an active enzyme without causing any problem in the body. Earlier, we have examined the possibility of MASP-3 activation within the LP (18); however, as we found out in this study, this is not the case. Hence, our goal was to decipher the activation mechanism MASP-3.

Although isolated MASP-3 does not autoactivate on its own, we did not exclude this possibility for PRM-associated MASP-3. Therefore, we used a catalytically inactive S664A variant of full-length MASP-3 (MASP-3–S/A) in our cleavage (“activation”) studies. Most assays were performed with this variant. We found that both fluorescently labeled and unlabeled MASP-3–S/A became cleaved in the blood using hirudin plasma as a model. The cleavage of labeled MASP-3–S/A was easily detectable at about the physiological concentration, and it occurred fairly rapidly (t1/2 ≈ 1–2 h); however only ∼30% of MASP-3–S/A–Cy5 was cleaved. Unlabeled MASP-3–S/A at ∼10-fold physiological concentration was activated slower (t1/2 ≈ 4–8 h), but a higher portion of MASP-3 was cleaved. In EDTA plasma the activation does not occur.

At this point, it was clear that MASP-3 activation occurs in the blood, and Ca2+ or another divalent cation is required for the activation. Initially we thought that this behavior might be attributed to the fact that MASPs dimerize and are associated with PRMs in a Ca2+-dependent fashion. We carried out a series of experiments and found this hypothesis to be false. Our data are consistent with a mechanism that neither MASPs nor FD nor any proteases inhibited by C1-inhibitor are responsible for MASP-3 activation. Interestingly, the general serine-protease inhibitor Pefabloc only partially inhibited the MASP-3 activating enzyme, and the activating enzyme appeared to be heat labile.

The only efficient narrow-specificity inhibitor that completely prevented MASP-3 activation was the general PBA-PC inhibitor, dec-RVKR-cmk. All our observations are consistent with a mechanism that MASP-3 is activated in the blood by at least one PBA-PC. These enzymes are Ca2+-dependent and often heat-labile (36) serine proteases and are inhibited only poorly by general serine protease inhibitors, like Pefabloc. Partial cleavage of MASP-3–S/A–Cy5 is probably caused by preferential labeling on the lysine residue at cleavage site (Lys-Arg↓Ile). With all of our observations explained, the main question is which PBA-PCs are present in the blood?

One of the PBA-PCs, PCSK6 (PACE4), has been shown to activate corin, a protease responsible for the production of natriuretic peptides (22, 23). Interestingly, corin bears an activation loop very similar to that of MASP-3. Using PCSK6-knockout mice, it was also proved in vivo that PCSK6 is required for corin activation, and this process occurs extracellularly. PCSK6 is a secreted enzyme, which is activated by autoactivation either intracellularly or at the surface of cells (40, 41).

Based on our results and published data, we can say with a high probability that PCSK6 is the major MASP-3 activator in the blood. In contrast, other members of the proprotein convertase family can potentially serve as backup or contributing enzymes to MASP-3 maturation, as PBA-PCs have a high redundancy in their substrate specificity. Enzymes like furin (PCSK3) and PCSK7 are membrane-bound proteases, but they can enter the bloodstream by shedding. The presence of furin has actually been detected in the blood, although at a very low concentration (∼1 ng/ml) (42). The secreted isoform of PCSK5, termed PC5A or PC6A, has a very similar overall structure to PCSK6. In this respect, PCSK5 might also contribute to MASP-3 activation. In fact, our in vitro experiments proved that both PCSK5 and PCSK6 are capable of activating MASP-3. Based on their in vivo distribution, PCSK6 emerges as an MASP-3 activator in the blood, whereas PCSK5 might serve as an activator in the periphery.

In summary, we have uncovered the activation mechanism of MASP-3: it is activated by one or more PBA-PCs. Although further studies are required to determine the relative contribution of each potential candidate, PCSK6 is likely to be the major MASP-3 activator in the blood (Fig. 9). Proprotein convertases have been known to process complement components intracellularly (4345). Now, our discovery identifies a centrally important function of this enzyme class: extracellular activation of complement via the constitutive activation of MASP-3. This finding reveals a hitherto hidden essential link between these enzymes and the complement system.

We thank Júlia Balczer (Research Centre for Natural Sciences, Budapest, Hungary) for her excellent technical assistance and preparation of SGMI-1 and SGMI-2. The authors are grateful to Dr. Steffen Thiel (Aarhus University, Denmark) for providing rMBL and pEAK8-MASP-3.

This work was supported by National Research, Development and Innovation Office of Hungary (NKFIH) Grants KH130376 (Hungarian Scientific Research Fund [OTKA]), K119374 (OTKA), K119386 (OTKA), K134711 (OTKA), K135289 (OTKA), and 2017-1.2.1-NKP-2017-00002 (National Brain Research Program [NAP]). NKFIH Project 2018-1.2.1-NKP-2018-00005 has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the 2018-1.2.1-NKP funding scheme. This work was also supported by Hungarian Academy of Sciences Grant KEP5/2020.

J.D. and P.G. conceived and supervised the study. J.D. and G.O. wrote the manuscript. G.O., R.D., B.M.V., D.V., A.V.Á., and J.D. prepared the proteins and performed the experiments. P.G., G.P., and P.Z. revised the manuscript. G.P. developed the specific MASP inhibitors. H.F. provided C1-inhibitor-deficient plasma samples. All authors commented on the manuscript and agreed to its final version.

The online version of this article contains supplemental material.

Abbreviations used in this article:

aka

also known as

AP

alternative pathway

cf

catalytic fragment

CP

classical pathway

dec-RVKR-cmk

decanoyl-Arg-Val-Lys-Arg-chloromethylketone

D-GlcNAc

N-acetyl-d-glucosamine

FD

factor D

LP

lectin pathway

MASP

MBL-associated serine protease

MBL

mannose-binding lectin

NPGB

p-nitrophenyl-p′-guanidinobenzoate

PACE4

paired basic amino acid–cleaving enzyme 4

PBA-PC

paired basic amino acid–specific proprotein convertase

PRM

pattern recognition molecule

rMBL

recombinant human MBL

WB

Western blot

1
Dobó
,
J.
,
G.
Pál
,
L.
Cervenak
,
P.
Gál
.
2016
.
The emerging roles of mannose-binding lectin-associated serine proteases (MASPs) in the lectin pathway of complement and beyond.
Immunol. Rev.
274
:
98
111
.
2
Garred
,
P.
,
N.
Genster
,
K.
Pilely
,
R.
Bayarri-Olmos
,
A.
Rosbjerg
,
Y. J.
Ma
,
M. O.
Skjoedt
.
2016
.
A journey through the lectin pathway of complement-MBL and beyond.
Immunol. Rev.
274
:
74
97
.
3
Yongqing
,
T.
,
N.
Drentin
,
R. C.
Duncan
,
L. C.
Wijeyewickrema
,
R. N.
Pike
.
2012
.
Mannose-binding lectin serine proteases and associated proteins of the lectin pathway of complement: two genes, five proteins and many functions?
Biochim. Biophys. Acta
1824
:
253
262
.
4
Héja
,
D.
,
A.
Kocsis
,
J.
Dobó
,
K.
Szilágyi
,
R.
Szász
,
P.
Závodszky
,
G.
Pál
,
P.
Gál
.
2012
.
Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.
Proc. Natl. Acad. Sci. USA
109
:
10498
10503
.
5
Megyeri
,
M.
,
V.
Harmat
,
B.
Major
,
Á.
Végh
,
J.
Balczer
,
D.
Héja
,
K.
Szilágyi
,
D.
Datz
,
G.
Pál
,
P.
Závodszky
, et al
.
2013
.
Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.
J. Biol. Chem.
288
:
8922
8934
.
6
Degn
,
S. E.
,
L.
Jensen
,
A. G.
Hansen
,
D.
Duman
,
M.
Tekin
,
J. C.
Jensenius
,
S.
Thiel
.
2012
.
Mannan-binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway activation in human serum, whereas neither MASP-1 nor MASP-3 is required for alternative pathway function.
J. Immunol.
189
:
3957
3969
.
7
Ekdahl
,
K. N.
,
C.
Mohlin
,
A.
Adler
,
A.
Åman
,
V. A.
Manivel
,
K.
Sandholm
,
M.
Huber-Lang
,
K.
Fromell
,
B.
Nilsson
.
2019
.
Is generation of C3(H2O) necessary for activation of the alternative pathway in real life?
Mol. Immunol.
114
:
353
361
.
8
Forneris
,
F.
,
D.
Ricklin
,
J.
Wu
,
A.
Tzekou
,
R. S.
Wallace
,
J. D.
Lambris
,
P.
Gros
.
2010
.
Structures of C3b in complex with factors B and D give insight into complement convertase formation.
Science
330
:
1816
1820
.
9
Ricklin
,
D.
,
G.
Hajishengallis
,
K.
Yang
,
J. D.
Lambris
.
2010
.
Complement: a key system for immune surveillance and homeostasis.
Nat. Immunol.
11
:
785
797
.
10
Lesavre
,
P. H.
,
H. J.
Müller-Eberhard
.
1978
.
Mechanism of action of factor D of the alternative complement pathway.
J. Exp. Med.
148
:
1498
1509
.
11
Volanakis
,
J. E.
,
S. V.
Narayana
.
1996
.
Complement factor D, a novel serine protease.
Protein Sci.
5
:
553
564
.
12
Dahl
,
M. R.
,
S.
Thiel
,
M.
Matsushita
,
T.
Fujita
,
A. C.
Willis
,
T.
Christensen
,
T.
Vorup-Jensen
,
J. C.
Jensenius
.
2001
.
MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway.
Immunity
15
:
127
135
.
13
Dobó
,
J.
,
D.
Szakács
,
G.
Oroszlán
,
E.
Kortvely
,
B.
Kiss
,
E.
Boros
,
R.
Szász
,
P.
Závodszky
,
P.
Gál
,
G.
Pál
.
2016
.
MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked.
Sci. Rep.
6
:
31877
.
14
Pihl
,
R.
,
L.
Jensen
,
A. G.
Hansen
,
I. B.
Thøgersen
,
S.
Andres
,
F.
Dagnæs-Hansen
,
K.
Oexle
,
J. J.
Enghild
,
S.
Thiel
.
2017
.
Analysis of factor D isoforms in Malpuech-Michels-Mingarelli-Carnevale patients highlights the role of MASP-3 as a maturase in the alternative pathway of complement.
J. Immunol.
199
:
2158
2170
.
15
Banda
,
N. K.
,
S.
Acharya
,
R. I.
Scheinman
,
G.
Mehta
,
M.
Coulombe
,
M.
Takahashi
,
H.
Sekine
,
S.
Thiel
,
T.
Fujita
,
V. M.
Holers
.
2016
.
Mannan-binding lectin-associated serine protease 1/3 cleavage of pro-factor D into factor D in vivo and attenuation of collagen antibody-induced arthritis through their targeted inhibition by RNA interference-mediated gene silencing.
J. Immunol.
197
:
3680
3694
.
16
Hayashi
,
M.
,
T.
Machida
,
Y.
Ishida
,
Y.
Ogata
,
T.
Omori
,
M.
Takasumi
,
Y.
Endo
,
T.
Suzuki
,
M.
Sekimata
,
Y.
Homma
, et al
.
2019
.
Cutting edge: Role of MASP-3 in the physiological activation of factor D of the alternative complement pathway.
J. Immunol.
203
:
1411
1416
.
17
Oroszlán
,
G.
,
E.
Kortvely
,
D.
Szakács
,
A.
Kocsis
,
S.
Dammeier
,
A.
Zeck
,
M.
Ueffing
,
P.
Závodszky
,
G.
Pál
,
P.
Gál
,
J.
Dobó
.
2016
.
MASP-1 and MASP-2 do not activate pro-factor D in resting human blood, whereas MASP-3 is a potential activator: kinetic analysis involving specific MASP-1 and MASP-2 inhibitors.
J. Immunol.
196
:
857
865
.
18
Oroszlán
,
G.
,
R.
Dani
,
A.
Szilágyi
,
P.
Závodszky
,
S.
Thiel
,
P.
Gál
,
J.
Dobó
.
2017
.
Extensive basal level activation of complement mannose-binding lectin-associated serine protease-3: kinetic modeling of lectin pathway activation provides possible mechanism.
Front. Immunol.
8
:
1821
.
19
Seidah
,
N. G.
,
M.
Chrétien
.
1999
.
Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides.
Brain Res.
848
:
45
62
.
20
Plaimauer
,
B.
,
G.
Mohr
,
W.
Wernhart
,
M.
Himmelspach
,
F.
Dorner
,
U.
Schlokat
.
2001
.
‘Shed’ furin: mapping of the cleavage determinants and identification of its C-terminus.
Biochem. J.
354
:
689
695
.
21
Seidah
,
N. G.
,
A.
Prat
.
2012
.
The biology and therapeutic targeting of the proprotein convertases.
Nat. Rev. Drug Discov.
11
:
367
383
.
22
Chen
,
S.
,
P.
Cao
,
N.
Dong
,
J.
Peng
,
C.
Zhang
,
H.
Wang
,
T.
Zhou
,
J.
Yang
,
Y.
Zhang
,
E. E.
Martelli
, et al
.
2015
.
PCSK6-mediated corin activation is essential for normal blood pressure.
Nat. Med.
21
:
1048
1053
.
23
Chen
,
S.
,
H.
Wang
,
H.
Li
,
Y.
Zhang
,
Q.
Wu
.
2018
.
Functional analysis of corin protein domains required for PCSK6-mediated activation.
Int. J. Biochem. Cell Biol.
94
:
31
39
.
24
Dobó
,
J.
,
V.
Harmat
,
E.
Sebestyén
,
L.
Beinrohr
,
P.
Závodszky
,
P.
Gál
.
2008
.
Purification, crystallization and preliminary x-ray analysis of human mannose-binding lectin-associated serine protease-1 (MASP-1) catalytic region.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
64
:
781
784
.
25
Dobó
,
J.
,
V.
Harmat
,
L.
Beinrohr
,
E.
Sebestyén
,
P.
Závodszky
,
P.
Gál
.
2009
.
MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity.
J. Immunol.
183
:
1207
1214
.
26
Megyeri
,
M.
,
P. K.
Jani
,
E.
Kajdácsi
,
J.
Dobó
,
E.
Schwaner
,
B.
Major
,
J.
Rigó
Jr.
,
P.
Závodszky
,
S.
Thiel
,
L.
Cervenak
,
P.
Gál
.
2014
.
Serum MASP-1 in complex with MBL activates endothelial cells.
Mol. Immunol.
59
:
39
45
.
27
Paréj
,
K.
,
A.
Hermann
,
N.
Donáth
,
P.
Závodszky
,
P.
Gál
,
J.
Dobó
.
2014
.
Dissociation and re-association studies on the interaction domains of mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, provide evidence for heterodimer formation.
Mol. Immunol.
59
:
1
9
.
28
Héja
,
D.
,
V.
Harmat
,
K.
Fodor
,
M.
Wilmanns
,
J.
Dobó
,
K. A.
Kékesi
,
P.
Závodszky
,
P.
Gál
,
G.
Pál
.
2012
.
Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.
J. Biol. Chem.
287
:
20290
20300
.
29
Vorup-Jensen
,
T.
,
E. S.
Sørensen
,
U. B.
Jensen
,
W.
Schwaeble
,
T.
Kawasaki
,
Y.
Ma
,
K.
Uemura
,
N.
Wakamiya
,
Y.
Suzuki
,
T. G.
Jensen
, et al
.
2001
.
Recombinant expression of human mannan-binding lectin.
Int. Immunopharmacol.
1
:
677
687
.
30
GE Healthcare
.
2006
.
Amersham CyDye Mono-Reactive NHS Esters: Reagents for the Labelling of Biological Compounds with Cy Monofunctional Dyes.
GE Healthcare, Little Chalfont
,
United Kingdom
.
31
Zundel
,
S.
,
S.
Cseh
,
M.
Lacroix
,
M. R.
Dahl
,
M.
Matsushita
,
J. P.
Andrieu
,
W. J.
Schwaeble
,
J. C.
Jensenius
,
T.
Fujita
,
G. J.
Arlaud
,
N. M.
Thielens
.
2004
.
Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2.
J. Immunol.
172
:
4342
4350
.
32
Degn
,
S. E.
,
L.
Jensen
,
P.
Gál
,
J.
Dobó
,
S. H.
Holmvad
,
J. C.
Jensenius
,
S.
Thiel
.
2010
.
Biological variations of MASP-3 and MAp44, two splice products of the MASP1 gene involved in regulation of the complement system.
J. Immunol. Methods
361
:
37
50
.
33
Thomsen
,
T.
,
A.
Schlosser
,
U.
Holmskov
,
G. L.
Sorensen
.
2011
.
Ficolins and FIBCD1: soluble and membrane bound pattern recognition molecules with acetyl group selectivity.
Mol. Immunol.
48
:
369
381
.
34
Haurum
,
J. S.
,
S.
Thiel
,
H. P.
Haagsman
,
S. B.
Laursen
,
B.
Larsen
,
J. C.
Jensenius
.
1993
.
Studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein A and comparison with two other collectins: mannan-binding protein and conglutinin.
Biochem. J.
293
:
873
878
.
35
Varga
,
L.
,
J.
Dobó
.
2014
.
C1 inhibitor: quantification and purification.
Methods Mol. Biol.
1100
:
189
205
.
36
Sucic
,
J. F.
,
J. M.
Moehring
,
N. M.
Inocencio
,
J. W.
Luchini
,
T. J.
Moehring
.
1999
.
Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain.
Biochem. J.
339
:
639
647
.
37
Yang
,
S. F.
,
R. H.
Chou
,
S. J.
Lin
,
S. Y.
Li
,
P. H.
Huang
.
2019
.
Serum PCSK6 and corin levels are not associated with cardiovascular outcomes in patients undergoing coronary angiography.
PLoS One
14
:
e0226129
.
38
Skjoedt
,
M. O.
,
Y.
Palarasah
,
L.
Munthe-Fog
,
Y.
Jie Ma
,
G.
Weiss
,
K.
Skjodt
,
C.
Koch
,
P.
Garred
.
2010
.
MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation.
Immunobiology
215
:
921
931
.
39
Iwaki
,
D.
,
K.
Kanno
,
M.
Takahashi
,
Y.
Endo
,
M.
Matsushita
,
T.
Fujita
.
2011
.
The role of mannose-binding lectin-associated serine protease-3 in activation of the alternative complement pathway.
J. Immunol.
187
:
3751
3758
.
40
Nagahama
,
M.
,
T.
Taniguchi
,
E.
Hashimoto
,
A.
Imamaki
,
K.
Mori
,
A.
Tsuji
,
Y.
Matsuda
.
1998
.
Biosynthetic processing and quaternary interactions of proprotein convertase SPC4 (PACE4).
FEBS Lett.
434
:
155
159
.
41
Mayer
,
G.
,
J.
Hamelin
,
M. C.
Asselin
,
A.
Pasquato
,
E.
Marcinkiewicz
,
M.
Tang
,
S.
Tabibzadeh
,
N. G.
Seidah
.
2008
.
The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates.
J. Biol. Chem.
283
:
2373
2384
.
42
He
,
Y.
,
L.
Ren
,
Q.
Zhang
,
M.
Zhang
,
J.
Shi
,
W.
Hu
,
H.
Peng
,
Y.
Zhang
.
2019
.
Serum furin as a biomarker of high blood pressure: findings from a longitudinal study in Chinese adults.
Hypertens. Res.
42
:
1808
1815
.
43
Wong
,
M. J.
,
G.
Goldberger
,
D. E.
Isenman
,
J. O.
Minta
.
1995
.
Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA.
Mol. Immunol.
32
:
379
387
.
44
Oda
,
K.
1992
.
Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.
J. Biol. Chem.
267
:
17465
17471
.
45
Oda
,
K.
,
Y.
Misumi
,
Y.
Ikehara
,
S. O.
Brennan
,
K.
Hatsuzawa
,
K.
Nakayama
.
1992
.
Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease.
Biochem. Biophys. Res. Commun.
189
:
1353
1361
.

The authors have no financial conflicts of interest.

Supplementary data