Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

Complement is vital to host immunity (1) and an effector system that facilitates the elimination of invading pathogens, but it must be tightly controlled to avoid inflammation and tissue damage (2). The complement system is activated via three pathways (3). The classical pathway is initiated by binding of the C1 complex and its associated serine proteases, C1r and C1s, to ligands, such as Ag-bound Igs. The lectin pathway is initiated by the MASP-1 and MASP-2 proteases associated with lectins, such as mannose-binding lectin (4). The alternative pathway is constitutively active through a so-called “tickover” mechanism and is amplified when Factors D and B are activated following the binding of C3b to foreign surfaces (5). Once activated, all three pathways converge at C3 and progress to the formation of the membrane attack complexes (or C5b–C9) on a target membrane (6).

From a therapeutic perspective, selective inhibition of the different complement-activation pathways to attenuate disease would ideally be required to avoid compromising the immune status of patients. In this regard, the classical and lectin pathways present more suitable targets, given that the alternative pathway acts as an amplification loop for both of these pathways (7). Accordingly, the C1r/C1s and MASP-1/MASP-2 serine protease systems present as ideal targets to inhibit the classical and lectin pathways, respectively.

Previously, a novel 17-kDa C1s inhibitor, designated BD001 (which we have termed gigastasin in this article), derived from the giant Amazonian leech (Haementaria sp.) (8) was identified and found to inhibit the classical pathway. However, its role in the lectin pathway was not assessed. Gigastasin includes a highly acidic C terminus containing three sulfotyrosine (sY) residues, which is similar to the stretch of amino acids found in complement component C4, the primary substrate for C1s and MASP-2. This part of C4 has more recently been shown to bind to C1s (9) and MASP-2 (10) via highly positively charged areas on the protease surface, termed exosites (anion-binding exosites [ABEs]).

In this study, we have shown that gigastasin is a potent inhibitor of C1s and MASP-2, is less active against MASP-1, and is a poor inhibitor of C1r. We have further determined the structure of gigastasin in complex with its most preferred target, C1s, and demonstrated that the mechanism of inhibition involves contact with the activation loop, the active site, and the ABE of the protease. Our data reveal that contact with the ABE regions of C1s and MASP-2 is vital to its inhibitory activity. Finally, we used deposition assays to assess the effect of gigastasin. Interestingly, in contrast to our predictions from the protease-inhibition kinetics, these data reveal that activation of the lectin pathway was more potently inhibited by gigastasin than was classical pathway activation.

Z–K–SBzl, Boc–LGR–AMC, and Boc–VPR–AMC were purchased from Bachem. 4,4′-Dithiodidyridine was purchased from Sigma-Aldrich. Normal human serum (NHS) and C1q-depleted serum were from Complement Technology (Tyler, TX). Mannose-binding lectin (MBL)-deficient serum was from the Statens Serum Institut (Copenhagen, Denmark). Murine monoclonal anti-C4d Ab was from Quidel (San Diego, CA). Goat anti-mouse secondary Ab (Alexa Fluor 488 conjugate) was from Life Technologies. Human dermal microvascular endothelial cells (HMEC-1) were a kind gift from the Centers for Disease Control and Prevention (Atlanta, GA).

C1s used in the crystallization experiments was purified from expired human plasma (Australian Red Cross Blood Service) using a modified protocol (11, 12). Briefly, the expired human plasma was clotted with 20 mM CaCl2, and serum was isolated by centrifugation to remove the clot. The euglobulin fraction was precipitated by extensive dialysis in 10 mM Bis-Tris (pH 6.1), 5 mM CaCl2, and 0.05 mM 4-nitrophenyl 4-guanidinobenzoate hydrochloride. The euglobulin fraction was solubilized in the same buffer plus 0.15 M NaCl. The C1 complement complex (zymogen) was purified using IgG-Sepharose (GE Healthcare). C1 zymogen was activated on Mono S (GE Healthcare) at neutral pH in the absence of 4-nitrophenyl 4-guanidinobenzoate hydrochloride. Activation of C1 was monitored using reducing and nonreducing SDS-PAGE. The active C1 complex was dissociated into its subcomponents C1q, C1r, and C1s by dialysis in the presence of EDTA to remove calcium. The full-length active C1s was separated from the other C1 subcomponents using Mono Q (GE Healthcare).

Complete plasmin cleavage of plasma-derived C1s generates several short fragments from the N terminus (<15 kDa) and a disulfide-linked CCP1–CCP2–SP region of C1s (∼50 kDa, residues L285–D688) (13). To produce the plasmin-cleaved C1s for structural studies, full-length active C1s (1 mg/ml) was incubated with human plasmin (Haematologic Technologies) (enzyme/substrate ratio [w/w] 1:100) in 50 mM Tris-HCl, (pH 7.2), 0.15 M NaCl, and 20 mM EDTA at 37°C for 1 h. Cleavage of C1s was monitored using SDS-PAGE. The cleaved C1s mixture was used to make complexes with gigastasin.

The gigastasin gene was synthesized using the mature primary sequence reported (8) and codon optimized for expression in insect cells. The recombinant gene also has a honeybee melittin signal peptide for secretion, an N terminus 6x histidine tag for purification, and an enterokinase cleavage site for removal of the purification tag, if required. The gigastasin gene was cloned into the pFastBac1 vector for expression in High Five insect cells. The spent insect cell media containing the secreted recombinant gigastasin was dialyzed extensively in 20 m Tris-HCl (pH 8), 0.3 M NaCl before being loaded onto Ni Sepharose (GE Healthcare) equilibrated in the same buffer. The column was washed with the same buffer plus 40 mM imidazole. Gigastasin was eluted with the same buffer containing 0.5 M imidazole. The purified inhibitor was buffer exchanged into 20 mM Tris-HCl (pH 8), 0.3 M NaCl for storage at 4°C.

C1s (plasmin cleaved) was mixed with gigastasin in a molar ratio of 1:2 in 0.1 M Tris-HCl (pH 8), 0.3 M NaCl and incubated on ice for 30 min. The N-terminal fragments of C1s generated by plasmin cleavage, plasmin, and free C1s were removed using chromatography on Ni Sepharose. Eluted fractions were further purified using Mono Q to remove free gigastasin and C1s. The purification steps were monitored using SDS-PAGE. Fractions containing the C1s/gigastasin complex were pooled and concentrated by ultrafiltration to 5 mg/ml for crystallization.

Initial attempts to solve the crystal structure of the full-length C1s/gigastasin complex were unsuccessful. The protein complex crystallized readily, but the crystals diffracted poorly and were severely anisotropic. To obtain different crystal forms, the inhibitor was complexed with plasmin-truncated C1s, and this was used to set up further crystallization trials using the sparse-matrix approach with the hanging-drop vapor-diffusion technique at both 4 and 20°C. A single hit (0.1 M Bis-Tris, pH 5.5, 25% [w/v] PEG 3350) was obtained at 4°C after several months. The pyramid-shaped C1s/gigastasin complex crystals were cryoprotected with the addition of 10% (v/v) ethylene glycol and flash-frozen in liquid nitrogen.

Complete x-ray diffraction data of C1s/gigastasin complex were collected on the MX2 beamline at the Australian Synchrotron (Table II). Diffraction data were indexed, integrated, and scaled using MOSFLM (14) and SCALA (15) from the CCP4 package (16).

The complex structure was solved by molecular replacement [using PHASER from the CCP4 suite (17)] using the C1s SP domain (1ELV, residue 410–668) (18) as the probe in the space group P3121. Each asymmetric unit contains one single C1s/gigastasin complex. After the initial refinement [using REFMAC5 (19)] with the C1s SP domain, the additional densities of the missing CCP1 and CCP2 domains, as well as gigastasin, were clearly visible. The missing structures were built in manually using COOT (20), with refinement cycles using Phenix (21) or the CCP4 suite (16). The progress of refinement was monitored using the Rfree value, and the final refinement statistics are listed in Table II. Coordinates and structure factors were submitted to the Protein Data Bank (PDB) under ID 5UBM (https://www.rcsb.org/pdb/results/results.do?tabtoshow=Unreleased&qrid=4A0C606A).

Constructs for the recombinant expression of C1s, MASP-2, and MASP-1 CCP1–CCP2–SP segment were synthesized by GenScript and cloned into a pET-17b vector (GenScript, Piscataway, NJ). The CCP1–CCP2–SP segment of C1s, the C1s ABE mutant, MASP-2, the MASP-2 ABE mutant, and MASP-1 were produced as described previously (22). Mutagenesis of the synthesized cDNA for recombinant C1s CCP12SP (residues K281 to D688) was carried out as described previously (9) to introduce a cysteine residue at the N terminus of selected proteins. The sequences of all variants were confirmed by dsDNA sequencing. Expression, refolding, and purification of all proteins were carried out as described previously (9). Where required, C1s was activated prior to use by incubating overnight at room temperature (RT) with C1r, as previously described (9).

Multiple-site mutagenesis of MASP-2 CCP12SP (K450A, K503A, R578A, and R583A) mutants were carried out using a QuikChange Site-Directed Mutagenesis Kit, following the manufacturer’s methods, and using splice-overlap PCR. Sequences of variants were confirmed by dideoxy DNA sequencing. Expression, refolding, and purification of proteins were carried out as described previously (23).

The activity of C1s and the C1s ABE mutant was measured using the thioester substrate Z–K–SBzl (200 μM) and the chromogenic thiol reagent 4,4′ dithiodidyridine (1 mM) (24), with an increase in absorbance observed at 324 nm. The activity of MASP-1 was measured using the fluorescence quenched substrate Boc–VPR–AMC (50 μM; excitation: 360 nm, emission: 460 nm). The activity of MASP-2 and MASP-2 variants was measured using Boc–LGR–AMC (200 μM; excitation: 360 nm, emission: 460 nm).

All assays were conducted in 20 mM Tris, 100 mM NaCl, 0.005% (v/v) Triton X-100 (pH 7.4) at 37°C in a FLUOstar Omega plate reader (BMG Labtech). Gigastasin was serially diluted, and the residual protease activity was measured. Data were fitted to the Morrison Equation (25) using a fixed total enzyme concentration.

viv0=1([E]T+[I]T+Kiapp)([E]T+[I]T+Kiapp)24[E]T[I]T2[E]T

Apparent equilibrium constant for inhibition values (Kiapp) were converted to Ki using the following relationship:

Ki=Kiapp1+(SKM)

Surface plasmon resonance studies were performed using a Biacore T100. For these experiments, the N-terminal biotinylated and sulfated peptide, C12S3 (Biotin-EDPNEEsYEsYDsYE), and its nonsulfated counterpart (Biotin-EDPNEEYEYDYE) were captured by streptavidin on the Biacore SA chip. The peptide was synthesized essentially as described previously (26). Unused biotin-binding sites were blocked by free biotin (60 s injection of 10 mM biotin at 30 μl/min). For the peptide-immobilization strategy, peptides (380 μM) in water were injected over an SA sensor chip (GE Life Sciences) surface for 30 s at 5 μl/min, resulting in a final immobilization level of 419 RU. The activated forms of C1s CCP12SPS632A, C1s CCP12SPS632A, K575A, R576A, R581A, K583A, MASP-2S632A, and MASP-2S632A, R578A, R583A were injected at concentrations ranging from 0 to 1 μM of each protein for 60 s at 50 μl/min, with a 5-min dissociation, followed by regeneration with 10 mM glycine (pH 2) for 30 s at 30 μl/min and 2 M NaCl for 30 s at 30 μl/min to generate Biacore kinetic sensorgrams (Fig. 3). The experiment was repeated in triplicate for each protein, and sensorgrams from triplicate runs were superimposable. The observed maximal response unit for each concentration of enzyme was plotted against the concentration of enzyme used to yield curves (insets) shown in Fig. 3 that were fitted to a one-site, specific binding equation in GraphPad Prism 5.0, where the equation was as follows: R = Rmax × [E]/(Kd + [E]).

Cells were cultured between passages 17 and 20. HMEC-1 cells were cultured in MCDB131 nutrient medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 5 mM l-Glutamine (Life Technologies, Invitrogen), 10 ng/ml recombinant human EGF (R&D Systems), and 1 μg/ml hydrocortisone (Tocris Bioscience) in a humidified incubator (95% O2, 5% CO2) at 37°C. HMEC-1 cells were grown to 90% confluence, washed with PBS, and starved for 16 h in serum-free media. Cells were washed with PBS and lifted into suspension with Accutase (Innovative Cell Technologies), after which they were washed twice with PBS and suspended at a concentration of ∼10 × 106 cells per milliliter. Then they were incubated at 37°C for 1 h with 10% (v/v) NHS, C1q-depleted serum, MBL-deficient serum, or corresponding serum that was heated (1 h at 57°C) to inactivate complement, in the presence of various concentrations of gigastasin. Cells were gently centrifuged and resuspended in FACS buffer (PBS, containing 1% [w/v] BSA) supplemented with murine monoclonal anti-C4d Ab at 4 μg/ml for 1 h at 4°C. Cells were gently pelleted and resuspended in 100 μl of FACS buffer with polyclonal FITC-conjugated goat anti-mouse IgG Abs (1:400; ∼5 μg/ml) for 45 min at 4°C. The cells were finally resuspended in FACS buffer with 0.1 mg/ml propidium iodide and analyzed by flow cytometry using a FACS LSR system (BD Biosciences, Mountain View, CA), as previously described (27).

Unless stated otherwise, all incubations were carried out at RT, and each step was followed by four washes with 50 mM Tris-HCl, 150 mM NaCl, and 0.1% (v/v) Tween 20 (pH 8). Microtiter plates (MaxiSorp; Nunc) were coated with 5 μg/ml heat-aggregated human IgG, to activate the classical pathway, or with 100 μg/ml mannan, to activate the lectin pathway, in 75 mM Na2CO3 (pH 9.6) overnight at 4°C and subsequently blocked for 2 h with 1% (w/v) BSA in PBS (blocking solution). Various dilutions of gigastasin (or BSA) in GVB++ (5 mM veronal buffer [pH 7.35], 140 mM NaCl, 0.1% [w/v] gelatin, 0.15 mM CaCl2, 1 mM MgCl2) were preincubated with 2% (v/v) human serum for 15 min before adding to the plates and incubated for 20 min at 37°C. The amount of deposited C4b was detected with a specific rabbit polyclonal Ab against C4c (Dako), diluted 1:4000 in blocking solution, and incubated for 1 h on the plate. HRP-conjugated anti-rabbit IgG (Dako) was diluted 1:2000 in blocking solution and allowed to bind for 30 min before bound enzyme was quantified using 1,2-phenylenediamine dihydrochloride (OPD) tablets (Kem-En-Tec Diagnostics), and the absorbance was measured at 490 nm.

Gigastasin was expressed in insect cells using baculovirus and purified. The equilibrium-inhibitory constants for the complement proteases that are able to cleave peptide substrates were determined. The results revealed that C1s was most potently inhibited by gigastasin, followed closely by MASP-2 (2.4-fold higher Ki), whereas the inhibitor was 32-fold less inhibitory toward MASP-1 (Table I). The inhibitor only had an effect on the cleavage of C1s by the C1r protease of the classical pathway at very high concentrations, suggesting that it is a poor inhibitor of C1r (data not shown).

Table I.
Equilibrium inhibitory constants for interaction between gigastasin and complement-initiating proteases
ProteaseKi (nM; mean ± SE)
C1s 0.394 ± 0.024 
C1sK575A, R576A, R581A, K583A 273 ± 17 
MASP-2 0.952 ± 0.120 
MASP-2R578A, R583A 91.9 ± 15.6 
MASP-2K450A, K503A, R578A, R583A 803 ± 74 
MASP-1 12.6 ± 1.5 
ProteaseKi (nM; mean ± SE)
C1s 0.394 ± 0.024 
C1sK575A, R576A, R581A, K583A 273 ± 17 
MASP-2 0.952 ± 0.120 
MASP-2R578A, R583A 91.9 ± 15.6 
MASP-2K450A, K503A, R578A, R583A 803 ± 74 
MASP-1 12.6 ± 1.5 

Bioinformatics analysis suggested that gigastasin is a distantly related member of the antistasin superfamily of protease inhibitors (Fig. 1) (28). These data reveal that it also contains an additional sequence at its N terminus that appears unique with respect to antistasin-like inhibitors or any other serine protease inhibitor characterized to date. The analysis also reveals that the C terminus of gigastasin contains a number of negatively charged amino acids, including three sY residues. The interaction between the antithrombotic agent, hirudin, and thrombin is markedly strengthened by the binding of electronegative residues located on the C terminus of the inhibitor to the ABE of the enzyme (29, 30). Therefore, it is highly possible that the electronegative C-terminal “tail” of gigastasin might also be interacting with the ABE regions of the complement proteases, such as C1s and MASP-2, to augment inhibition.

FIGURE 1.

The structure of gigastasin. (A) An overview of the C1s/gigastasin complex. CCP1/2 is in purple, the C1s protease domain is in cyan, and gigastasin is in pink (N-terminal domain)/orange (antistasin domain)/green (C-terminal domain). The P1 residue (orange sticks) and sY sequence (green/yellow/red sticks) are shown. The N and C termini are labeled. (B) Sequence alignment of gigastasin with other members of the antistasin family of inhibitors. (C) Diagram showing gigastasin alone; coloring and labeling are as in (A), with disulfide bonds shown as a yellow stick. (D) Electrostatic potential surface map of the C1s protease domain, with gigastasin shown as the gray cartoon and the amino acids that interact with C1s in a stick representation. Key interaction points are labeled. Note the extensive positively charged exosite (green dashed circle) that makes up the sY binding site. (E) Electron density map of the loop of gigastasin that makes contact with the active site of C1s.

FIGURE 1.

The structure of gigastasin. (A) An overview of the C1s/gigastasin complex. CCP1/2 is in purple, the C1s protease domain is in cyan, and gigastasin is in pink (N-terminal domain)/orange (antistasin domain)/green (C-terminal domain). The P1 residue (orange sticks) and sY sequence (green/yellow/red sticks) are shown. The N and C termini are labeled. (B) Sequence alignment of gigastasin with other members of the antistasin family of inhibitors. (C) Diagram showing gigastasin alone; coloring and labeling are as in (A), with disulfide bonds shown as a yellow stick. (D) Electrostatic potential surface map of the C1s protease domain, with gigastasin shown as the gray cartoon and the amino acids that interact with C1s in a stick representation. Key interaction points are labeled. Note the extensive positively charged exosite (green dashed circle) that makes up the sY binding site. (E) Electron density map of the loop of gigastasin that makes contact with the active site of C1s.

Close modal

To understand the structural basis for gigastasin inhibition, we determined the 2.5-Å x-ray crystal structure of the complex between the CCP1–CCP2–SP fragment of plasma-derived C1s and gigastasin (Fig. 1, Table II). The structure revealed that gigastasin consists of a three-domain protein (Fig. 1C) that binds to C1s, with a total buried surface area of 1365.6 Å2. The central portion of the inhibitor (59–91) adopted the predicted antistasin-like fold and binds into the protease active site in a canonical fashion (Fig. 1A, 1E; buried surface area = 788.2 Å2). The primary specificity residue of gigastasin, R65 (P1), docks into the S1 specificity pocket of the enzyme (Fig. 1D, 1E) and forms extensive interactions at the base of the pocket (including a salt bridge to D626). However, it is also notable that gigastasin completely blocks all other subsites of C1s and forms contacts that span from P6 to P4′ (Fig. 2A).

Table II.
Crystallographic statistics

 

 
Data collection  
 Temperature (°K) 100 
 Space group P3121 
 Unit cell parameters (Å)  
  a, b 89.35 
  c 146.87 
 Resolution (Å) 53.27–2.50 (2.64–2.50) 
 Completeness (%) 100 (100) 
 Total reflections 209,827 (31,050) 
 Unique reflections 24,136 (3,480) 
 Multiplicity 8.7 (8.9) 
 Rpim (%) 5.3 (36.0) 
 Mean I/σ (I) 10.8 (2.1) 
 Mosaicity (°) 0.56 
Refinement  
 Nonhydrogen atoms 3,936 
 C1s atoms 2,940 
 Inhibitor atoms 870 
 Water 126 
 Resolution (Å) 41.37–2.50 
 Rfactor (%) 17.67 
 Rfree (%) 22.23 
 Root-mean-square deviation from ideality  
  Bond lengths (Å) 0.004 
  Bond angles (Å) 0.872 
  Chirality (°) 0.036 
  Planarity (°) 0.004 
  Dihedrals (°) 14.253 
 Ramachandran plot (%)  
  Preferred regions 95.84 
  Allowed regions 4.16 
  Disallowed regions 0.00 
 B factors (Å2 
  Average all atoms 56.89 
  Average C1s 49.96 
  Average inhibitor 80.32 
 Average water 45.22 

 

 
Data collection  
 Temperature (°K) 100 
 Space group P3121 
 Unit cell parameters (Å)  
  a, b 89.35 
  c 146.87 
 Resolution (Å) 53.27–2.50 (2.64–2.50) 
 Completeness (%) 100 (100) 
 Total reflections 209,827 (31,050) 
 Unique reflections 24,136 (3,480) 
 Multiplicity 8.7 (8.9) 
 Rpim (%) 5.3 (36.0) 
 Mean I/σ (I) 10.8 (2.1) 
 Mosaicity (°) 0.56 
Refinement  
 Nonhydrogen atoms 3,936 
 C1s atoms 2,940 
 Inhibitor atoms 870 
 Water 126 
 Resolution (Å) 41.37–2.50 
 Rfactor (%) 17.67 
 Rfree (%) 22.23 
 Root-mean-square deviation from ideality  
  Bond lengths (Å) 0.004 
  Bond angles (Å) 0.872 
  Chirality (°) 0.036 
  Planarity (°) 0.004 
  Dihedrals (°) 14.253 
 Ramachandran plot (%)  
  Preferred regions 95.84 
  Allowed regions 4.16 
  Disallowed regions 0.00 
 B factors (Å2 
  Average all atoms 56.89 
  Average C1s 49.96 
  Average inhibitor 80.32 
 Average water 45.22 
FIGURE 2.

Details of the major contacts made between gigastasin and C1s. (A) The inhibitor makes extensive canonical interactions from P6 to P4′. D60 (P6) forms a hydrogen bond to Y610. F62 (P4) forms π-stacking interactions with the side chain of Y610 and also packs against P657. The main chain carbonyl of K63 (P3) forms a hydrogen bond to the backbone amide of G656. C64 (P2) forms a disulfide bond with C83 and is loosely packed into the shallow S2 pocket. The side chain of R65 (P1) forms a buried salt bridge with D626 in the S1 pocket. The carbonyl oxygen of the P1 further forms the anticipated interactions with the oxyanion hole (formed from backbone amides of G630, D631, and S632 [catalytic serine (S195 in chymotrypsin numbering)]; only S632 is labeled). On the prime side, L66 (P1′) is positioned such that it makes van der Waals interactions with A460, V476, and P458 (these latter three residues are not shown). G67 (P2′) and C68 (P3′) form main-chain hydrogen bonds with the active site; finally, the side chain of T69 (P4′) interacts with the side chain of N457. (B) Additional interactions made by gigastasin include W17 forming a hydrogen bond with Y662 and the carbonyl oxygen of C54 interacting with the side chain of Q658. The C-terminal sY residues Y117 and Y119 both interact with Q493 and form ionic contacts with R576, R581, and K583.

FIGURE 2.

Details of the major contacts made between gigastasin and C1s. (A) The inhibitor makes extensive canonical interactions from P6 to P4′. D60 (P6) forms a hydrogen bond to Y610. F62 (P4) forms π-stacking interactions with the side chain of Y610 and also packs against P657. The main chain carbonyl of K63 (P3) forms a hydrogen bond to the backbone amide of G656. C64 (P2) forms a disulfide bond with C83 and is loosely packed into the shallow S2 pocket. The side chain of R65 (P1) forms a buried salt bridge with D626 in the S1 pocket. The carbonyl oxygen of the P1 further forms the anticipated interactions with the oxyanion hole (formed from backbone amides of G630, D631, and S632 [catalytic serine (S195 in chymotrypsin numbering)]; only S632 is labeled). On the prime side, L66 (P1′) is positioned such that it makes van der Waals interactions with A460, V476, and P458 (these latter three residues are not shown). G67 (P2′) and C68 (P3′) form main-chain hydrogen bonds with the active site; finally, the side chain of T69 (P4′) interacts with the side chain of N457. (B) Additional interactions made by gigastasin include W17 forming a hydrogen bond with Y662 and the carbonyl oxygen of C54 interacting with the side chain of Q658. The C-terminal sY residues Y117 and Y119 both interact with Q493 and form ionic contacts with R576, R581, and K583.

Close modal

In comparison with other members of the antistasin superfamily, gigastasin includes unique N- and C-terminal domains. The N-terminal domain (3–58) includes five disulfide bonds. Despite representing the largest domain, this region only makes two significant contacts with C1s (buried surface area = 265.9 Å2). Most notably, W17 forms extensive van der Waals contacts with the N-terminal (activation loop) region of C1s (Fig. 2B). In addition, the backbone carbonyl of C54 forms a hydrogen bond to Q658 on the C1s protease domain.

Similarly, the C-terminal domain (92–119) of gigastasin forms relatively limited contacts (buried surface area = 332.1 Å2) with the protease domain. These contacts include a salt bridge between R109 and D577 on the C1s serine protease domain. Most notably, however, the far C-terminal sequence includes two sY residues that directly contact the ABE of C1s. Major polar interactions made in these regards include contact with Q493, R576, R581, and K583 (Fig. 2B). These latter three residues are known to be the major features of the ABE found on the surface of C1s (9, 22).

Because the electron density for the amino acids from the C terminus of gigastasin interacting with the ABE of C1s was not completely clear, we further investigated the importance of this interaction in vitro. To do this, we used a mutant of C1s with the 4 aa constituting the ABE of this enzyme mutated to alanine residues (C1sK575A, R576A, R581A, K583A) (9, 22). Two mutants of MASP-2 were used: one in which the two residues that were shown to be vital for interactions with C4 were mutated to alanine residues (MASP-2R578A, R583A) (10) and the other in which all four residues originally predicted to be interacting with C4 were mutated to alanine residues (MASP-2K450A, K503A, R578A, R583A) (10). The Ki for gigastasin with the ABE mutant of C1s was 693-fold weaker than the wild-type enzyme (Table I), indicating that the interaction between the C terminus of gigastasin and the ABE is indeed of major importance to this enzyme. In the case of MASP-2, the double mutant form (MASP-2R578A, R583A) was inhibited 97-fold less effectively, whereas the mutant in which all 4 aa of the ABE were mutated was inhibited 843-fold less effectively, indicating that, for this enzyme, the entire ABE was required for full interaction with gigastasin (Table I).

To further delineate the details of the interaction with the ABE, a 12-aa peptide representing the C-terminal tail of gigastasin, C12S3, was synthesized bearing intact sulfation of the tyrosine residues (31). The synthetic peptide was modified with an N-terminal biotin residue to allow immobilization to a streptavidin-coated surface. Measurement of the binding kinetics of C1s and MASP-2, as well as the full ABE mutant of C1s and the mutant of MASP-2 in which two residues of the ABE were mutated, was carried out using surface plasmon resonance. This showed that wild-type C1s bound to the immobilized peptide with a Kd value of 810 nM, whereas MASP-2 had a 16-fold greater affinity for the peptide (Kd = 52 nM) (Fig. 3). The mutant forms did not display any binding to the molecule. The wild-type molecules also bound a nonsulfated form of the peptide with significantly lowered affinity (results not shown), indicating that the sulfation was important for the reaction.

FIGURE 3.

Analysis of the interaction between complement proteases and gigastasin C-terminal peptide. Sensorgrams from surface plasmon resonance measurements for the indicated amounts of C1s and MASP-2 flowed over peptide Biotin-EDPNEEsYEsYDsYE attached to streptavidin immobilized to the chip. The experiments were conducted in triplicate and showed excellent overlap. Insets: the response units obtained at equilibrium for each concentration of protease were plotted against the respective protease concentration and fitted using a one site, specific binding model on GraphPad Prism (regression coefficients for the fits = 0.99) to yield the Kd values shown.

FIGURE 3.

Analysis of the interaction between complement proteases and gigastasin C-terminal peptide. Sensorgrams from surface plasmon resonance measurements for the indicated amounts of C1s and MASP-2 flowed over peptide Biotin-EDPNEEsYEsYDsYE attached to streptavidin immobilized to the chip. The experiments were conducted in triplicate and showed excellent overlap. Insets: the response units obtained at equilibrium for each concentration of protease were plotted against the respective protease concentration and fitted using a one site, specific binding model on GraphPad Prism (regression coefficients for the fits = 0.99) to yield the Kd values shown.

Close modal

Activation of the early stages of complement can be monitored by measuring the deposition of C4 cleavage products (C4b or C4d) on cellular surfaces. Activation within NHS will monitor activation by all pathways. In this context, gigastasin was able to inhibit C4d deposition by ∼75% at a concentration of 1 μM, whereas 500 nM gigastasin decreased C4d deposition by ∼50% (Fig. 4). Assaying in the presence of serum depleted of MBL, a primary recognition complex of the lectin pathway, provided an estimate of the activation occurring via the classical pathway. Gigastasin was able to inhibit activation via this pathway by ∼50% at a concentration of 1.25 μM, whereas 5 μM of the inhibitor was required to decrease complement activation via this pathway by >80%. Assays in the presence of C1q-depleted serum gave a measure of the activation occurring via the lectin pathway: only 0.5 μM gigastasin was required to inhibit activation by >85%, whereas 0.06 μM gigastasin resulted in >50% inhibition of the activation of the lectin pathway. Our findings that, in this cellular system, gigastasin favors the lectin pathway is somewhat surprising, given the preceding biochemical data. Therefore, we sought validation by a second ELISA approach in which we quantified the deposition of C4b on surfaces required for activation of the different pathways in the presence and absence of gigastasin (Fig. 4D). These experiments also revealed a more prominent inhibitory effect of gigastasin on the lectin pathway compared with the classical pathway.

FIGURE 4.

Gigastasin inhibits the classical and lectin complement pathways in a dose-dependent manner. (A) Complement pathways were activated by incubating HMEC-1 cells with 10% (v/v) NHS. In a similar manner, HMEC-1 cells were incubated with 10% (v/v) MBL-deficient serum or C1q-depleted serum to reflect activation of primarily the classical (B) or lectin (C) complement pathway, respectively, prior to measuring C4d deposition by flow cytometry. C4d-staining graphs (right panels) show FITC-conjugated anti-C4d fluorescence (y-axis) and cell count (x-axis) for the NHS positive control (purple), background fluorescence (orange; derived from values for heat-inactivated serum), buffer negative control (blue), and incubation conducted with gigastasin (red). Bar graphs (left panels) represent the average mean fluorescence intensity (MFI) in the presence of various gigastasin concentrations. A volume of the buffer used to dissolve gigastasin equivalent to the highest gigastasin concentration tested was used as a negative control. The average MFI for serum-activated samples was corrected for background fluorescence by subtracting the average MFI value derived from the corresponding heat-inactivated serum samples. Values are MFI ± SEM of duplicate samples and are representative of two independent experiments. (D) Gigastasin inhibits C4b deposition. Heat-aggregated human IgG (classical pathway) or mannan (lectin pathway) was immobilized and allowed to activate human serum containing various concentrations of gigastasin or BSA as a negative control (preincubated for 15 min at RT). After 20 min of incubation at 37°C, the plates were washed, and the deposited C4b was detected with a specific polyclonal Ab. The absorbance obtained in the absence of gigastasin was defined as 100%. The average of three independent experiments performed in duplicate is presented, with error bars indicating SD.

FIGURE 4.

Gigastasin inhibits the classical and lectin complement pathways in a dose-dependent manner. (A) Complement pathways were activated by incubating HMEC-1 cells with 10% (v/v) NHS. In a similar manner, HMEC-1 cells were incubated with 10% (v/v) MBL-deficient serum or C1q-depleted serum to reflect activation of primarily the classical (B) or lectin (C) complement pathway, respectively, prior to measuring C4d deposition by flow cytometry. C4d-staining graphs (right panels) show FITC-conjugated anti-C4d fluorescence (y-axis) and cell count (x-axis) for the NHS positive control (purple), background fluorescence (orange; derived from values for heat-inactivated serum), buffer negative control (blue), and incubation conducted with gigastasin (red). Bar graphs (left panels) represent the average mean fluorescence intensity (MFI) in the presence of various gigastasin concentrations. A volume of the buffer used to dissolve gigastasin equivalent to the highest gigastasin concentration tested was used as a negative control. The average MFI for serum-activated samples was corrected for background fluorescence by subtracting the average MFI value derived from the corresponding heat-inactivated serum samples. Values are MFI ± SEM of duplicate samples and are representative of two independent experiments. (D) Gigastasin inhibits C4b deposition. Heat-aggregated human IgG (classical pathway) or mannan (lectin pathway) was immobilized and allowed to activate human serum containing various concentrations of gigastasin or BSA as a negative control (preincubated for 15 min at RT). After 20 min of incubation at 37°C, the plates were washed, and the deposited C4b was detected with a specific polyclonal Ab. The absorbance obtained in the absence of gigastasin was defined as 100%. The average of three independent experiments performed in duplicate is presented, with error bars indicating SD.

Close modal

The leech molecule previously designated BD001 was first described as an inhibitor of the classical pathway enzyme, C1s, some time ago (8), but its mechanism of action and effects on the lectin pathway of complement have not been fully determined. We (9) and others investigators (32) have described the involvement of ABEs located on the surfaces of C1s and MASP-2 in the binding and cleavage of their primary substrate, C4. We were intrigued to note that the leech molecule contained a highly negatively charged C-terminal “tail” encompassing three sY residues, which is very similar to the situation found for the ABE-binding region of C4 (9, 32). This led us to hypothesize that the C-terminal tail of the leech molecule might play a role in binding to the ABE of C1s. This would be similar to the role of the negatively charged C-terminal region of hirudin, which binds to the ABE of thrombin and, thus, plays a crucial role in inhibiting the enzyme (29, 30).

Bioinformatic analysis revealed that the central inhibitor domain of the leech molecule was homologous to members of the antistasin family of protease inhibitors. This result, together with its derivation from the giant Amazonian leech, led us to propose the name gigastasin for this molecule. Intriguingly, the sequence alignments also revealed that the N terminus of the molecule bore no relation to other protease inhibitors, suggesting that it may play a hitherto unknown role in the mechanism of action of this inhibitor.

Solution of the structure of gigastasin in complex with C1s has given us our first insights into the detailed interactions that are made between an inhibitor and the active site of C1s. In addition, the structural and biochemical work carried out in this study has validated the hypothesis that the acidic tail of gigastasin does indeed bind to the ABE of C1s and plays a major role in the interaction between the inhibitor and this enzyme. This first visualization of the interaction between the sY residue and the ABE of C1s is particularly interesting because it is notably very similar to what might be expected for the interaction between the negatively charged region of C4 and the ABE of C1s. Finally, the N-terminal region of gigastasin has been shown to house an entirely novel interaction with the activation loop of C1s. This intriguing mechanism ensures that full inhibition of the protease only occurs with the activated form of the enzyme, because the point of interaction on the enzyme is only exposed upon activation (22).

Gigastasin, untested for its ability to inhibit lectin pathway proteases to date, was shown to have a slight preference for inhibition of C1s, but it was also an effective inhibitor of MASP-2 and, to a lesser extent, MASP-1. These data laid the basis for further work investigating the ability of the inhibitor to effect activation of the lectin pathway of complement. We used the gigastasin–C1s structure in superposition experiments to help explain the selectivity observed with regard to the different complement-initiation proteases. Superposition of gigastasin–C1s with the structure of MASP-2 (PDB ID: 1Q3X) (33) suggests that the inhibitor could interact with MASP-2 in an essentially identical fashion (i.e., we observed no predicted steric clashes) (data not shown). In contrast, a similar analysis of the gigastasin–C1s structure with respect to MASP-1 (PDB ID: 3GOV) (34) reveals that the latter protease would not be anticipated to be able to interact with the gigastasin N- and C-terminal domains in a similar fashion to C1s (Fig. 5). For example, N451 and F593 of MASP-1 are predicted to block the interactions made by W17 in gigastasin with either C1s or MASP-2. However, we note that the central antistasin-like domain of gigastasin would still be anticipated to bind to the active site of MASP-1 without significant steric clashes. Finally, in C1r (PDB ID: 1MD8) (35), the 491 loop of the enzyme blocks binding of the central antistasin family domain to the active site of the enzyme, and the other major interactions with the N and C termini of the inhibitor are also not predicted to occur, explaining why this protease is poorly inhibited (Fig. 5).

FIGURE 5.

Superimposition of the structures of MASP-1 (A) and Clr (B) onto the structure of gigastasin–C1s. (A) The side chains of N451 and F593 of MASP-1 (light pink, 3GOV) form significant steric clashes with W17 in gigastasin (gray). The equivalent residues in Cls (G440 and E574, respectively) form part of the binding pocket that interacts with inhibitor residue W17. (B) Clr (orange, 1MD8) 491 loop (Pro489-His492, stick model) forms close steric clashes with gigastasin (gray, C83-C85). The equivalent loop region (G479-E482) in C1s (highlighted as a stick model in cyan) does not interfere with gigastasin binding.

FIGURE 5.

Superimposition of the structures of MASP-1 (A) and Clr (B) onto the structure of gigastasin–C1s. (A) The side chains of N451 and F593 of MASP-1 (light pink, 3GOV) form significant steric clashes with W17 in gigastasin (gray). The equivalent residues in Cls (G440 and E574, respectively) form part of the binding pocket that interacts with inhibitor residue W17. (B) Clr (orange, 1MD8) 491 loop (Pro489-His492, stick model) forms close steric clashes with gigastasin (gray, C83-C85). The equivalent loop region (G479-E482) in C1s (highlighted as a stick model in cyan) does not interfere with gigastasin binding.

Close modal

Following the deciphering of the mechanism of action for gigastasin and understanding that it is a potent inhibitor of lectin pathway proteases in addition to C1s, we investigated the ability of the inhibitor to effect activation of the different complement pathways. As expected, and as previously described, gigastasin inhibited activation of the classical pathway (8) and did not effect activation via the alternative pathway using a cellular model of complement activation. Somewhat surprisingly, gigastasin was a far more potent inhibitor of the lectin pathway than the in vitro protease-inhibition results might have suggested. Because this was a surprising result, and noting that the MBL-deficient serum would still contain ficolin complexes that would be capable of activating the lectin pathway, we confirmed these results using a C4b-deposition assay on surfaces containing ligands that selectively activate each of the pathways of complement. Data from these assays confirmed the results obtained using the cellular assays. C1s, and thus the classical pathway, might be suggested to be the primary target of gigastasin based on the inhibitory constants obtained for interaction with the complement proteases; however, when one considers that the Ki value for MASP-2 is only 2.4-fold greater than that for C1s and that the inhibitor also targets MASP-1 to a reasonable degree, it is likely that inhibition of both activating proteases makes the lectin pathway much more susceptible to inhibition by gigastasin. Therefore, the absence of inhibition of C1r, the primary activating enzyme for the classical pathway, would be envisaged to outweigh the slightly more potent inhibition of C1s in terms of the effect of the inhibitor on the activation of these two pathways. The lower concentration of MASP-2 than C1s in the blood (36) might also ensure that inhibition of the lectin pathway is more efficient, although the excess of gigastasin used in the present experiments should have taken this concern into account. Overall, these results suggest that gigastasin is, in fact, more specific for the lectin pathway.

In summary, this study demonstrates that the leech-derived inhibitor, gigastasin, has a novel mechanism of action in which it targets the complement proteases C1s and MASP-2 using three regions of interaction. This novel member of the antistasin-like family of protease inhibitors shows great promise as a scaffold for the production of molecules that are specifically able to target each of the lectin and classical pathways of the complement system and, thus, ameliorate the role of these pathways in specific inflammatory diseases associated with these pathways.

This work was supported by National Health and Medical Research Council of Australia Project Grant 1082090 (to R.N.P.).

The coordinates and structure factors presented in this article have been submitted to the Protein Data Bank (https://www.rcsb.org/pdb/results/results.do?tabtoshow=Unreleased&qrid=4A0C606A) under accession number 5UBM.

Abbreviations used in this article:

ABE

anion-binding exosite

MBL

mannose-binding lectin

NHS

normal human serum

PDB

Protein Data Bank

RT

room temperature

sY

sulfotyrosine.

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The authors have no financial conflicts of interest.