Cell culture and protein expression
Complement component C2 (NM_000063) was cloned from a liver cDNA library (Invitrogen, Carlsbad, CA), and inserted into a plasmid carrying a collagen promoter (pX804) or into pCep4 (Invitrogen, Carlsbad, CA). HT1080 cells (CCL-121, ATCC, Manassas, VA) were grown and maintained in CD media (50% CD-CHO and 50% CD-293) (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2 incubator. Stable transfections were carried out using electroporation (450 V with 250 μF of capacitance and 30 μg of plasmid DNA suspended in 750 μl 1× PBS). 293-F cells (Invitrogen, Carlsbad, CA) were grown in Freestyle 293 media (Invitrogen, Carlsbad, CA) containing GlutaMAX. Cells were transiently transfected using a polyethylenimine (25 kD, Polysciences, Warrington, PA) to DNA ratio of 3:1 and were supplemented with 2.5 mg/ml Primatone (MP Biomedicals, Solon, OH) at 24 hours. Seventy-two hours after transfection conditioned media enriched in rhC2 was harvested from a 25 liter wave bioreactor (GE Healthcare, Piscataway, NJ) and then processed for purification.
Protein purification and biochemical characterization
A two-step purification process was developed for rhC2. First a SP Sepharose Fast Flow resin column (GE Healthcare, Piscataway, NJ) was used to capture the protein from harvest medium. The binding capacity of the SP resin was experimentally determined by ELISA to be 0.37 mg rhC2 per ml of SP resin. Protein was then eluted from the column and samples from collected fractions were analyzed by BCA Protein Assay Kit, (Pierce, Rockford, IL) to determine protein concentration and visualized by Coomassie staining (GelCode Blue Stain Reagent, Pierce, Rockford, IL) of 8-16% tris-glycine SDS-PAGE gels under reducing conditions (Invitrogen, Carlsbad, CA). The fractions containing full length rhC2 (102 kDa) were pooled and further purified over a Heparin Sepharose Fast Flow resin column (GE Healthcare, Piscataway, NJ). A binding capacity of 1 mg of total protein by BCA assay per ml of Heparin resin was used. Eluted fractions were assessed as described above and those containing rhC2 were pooled, concentrated and buffer-exchanged into storage buffer (10× PBS diluted to 1× in water and adjusted to pH 7). Forty five liters of harvest media from the WAVE bioreactor yielded 228 mg of purified rhC2. The resultant rhC2 protein was confirmed by N-terminal sequencing (Mayo Proteomics Research Center, Mayo Clinic College of Medicine in Rochester, MN) and was assessed for purity using size exclusion chromatography. Purified rhC2 (5 μg) was separated on a size exclusion chromatography (SEC) column (Tosoh SuperSW 3000, 10,000-500,000 Da, Tosoh, Montgomery, PA) with 25 mM sodium phosphate, pH 6.5 and 500 mM sodium chloride in MilliQ water as the mobile phase with a flow rate of 0.15 ml/min. The chromatogram was quantified using Exponential Skim Integration of the Empower software (Waters, Milford, MA). Using this procedure, the resultant rhC2 was determined to be 96% pure. The molecular weight and hydrodynamic radius of the major peak seen on size exclusion chromatography for rhC2 was analyzed using a multi angle static and dynamic light scattering system from Wyatt Technologies (Santa Barbara, CA) in conjunction with size exclusion chromatography method described above with the following modifications: the pH of the light scattering mobile phase buffer was increased to pH 7.0, the flow rate was increased to 0.3 mL/min, and 10 μg of rhC2 diluted (1:10) in light scattering mobile phase buffer.
Glycodigestion was performed to determine the presence of carbohydrates and sialic acid. Two micrograms of rhC2 was used for glycodigestion, for carbohydrate determination the protein was incubated for 1 hour and overnight at 37°C with Peptide N-glycosidase in reaction buffer (New England Biolabs, Beverly, MA). For sialic acid determination, rhC2 was incubated for 1 hour at 37°C with neuraminidase in reaction buffer (New England Biolabs, Beverly, MA). SDS-PAGE was performed with 250 ng of sample digested with above enzymes, loaded on 8-16% gel and visualized by Western blot. Thermal stability of rhC2 was assessed with differential scanning calorimetry using a MicroCal VP-DSC (GE Healthcare, Piscataway, NJ) with 400 μl buffer (25 mM sodium phosphate, 500 mM sodium chloride) per buffer/sample pairs in 96 deep well plates loaded in the CapVP-DSC autoloader. Data were collected with the calorimeter scanning from 10 to 100°C at a rate of 60°C/hour.
A sandwich ELISA was developed to allow specific quantitation of rhC2 during purification. The wells of a 96-well Nunc Immuno plate (Nalge Nunc International, Rochester, NY) were coated with 100 μl of 1 μg/ml anti-human C2 goat polyclonal antibody (R&D systems, Minneapolis, MN) in 50 mM sodium bicarbonate, pH 9.6. The plate was sealed and incubated overnight at RT. The next morning the wells were washed 3 times with ELISA Wash Buffer (0.1% Tween-20 in PBS), then 300 μl of Blocking Buffer (PBS, 0.05% Tween-20, 2% BSA) were added to each well and the plate was incubated for 1 hour at 37°C. Following the incubation, blocking buffer was removed, 100 μl volume of samples and standards (pre-diluted in blocking buffer) were added to the plate, and the plate was incubated for 1 hour at 37°C. Following incubation, the plate was washed as described above, the secondary antibody, a mouse anti-human C2 monoclonal antibody (Abcam, Cambridge, MA), was added to all wells at a final concentration of 1 μg/mL (100 μl per well) and the plate was incubated for 1 hour at 37°C then washed. After washing, 100 μl of 100 ng/ml of HRP-conjugated anti-mouse antibody (Promega, Madison, WI) was added to each well and the plate was incubated for 1 hour at 37°C then washed. Finally, 100 μl of the complete Peroxidase EIA Substrate Kit (BioRad, Hercules, CA) was added to each well and the plate incubated for 20 min at 37°C. The reaction was stopped by adding 100 μl of 2 N H2SO4 to each well and the plate was read at 450 nm on a Molecular Devices plate reader equipped with SoftMax Pro software. The standard curve had a dynamic range from 200 ng/ml to 3 ng/ml prepared by serially diluting C2 purified from human serum (R&D systems, Minneapolis, MN).
Cleavage of rhC2 by C1s
rhC2 and purified C2 from human plasma at a concentration of 25 μg/ml and 50 μg/ml, respectively were incubated with active C1s for 1 h at 37°C in veronal buffered saline with 0.15 mM Ca2+ and 0.5 mM Mg2+ (VBSCaMg) at a concentration of 70 μg/ml to induce complete cleavage of C2. C1s was produced and purified as described by Arlaud G.J. et al, . The proteins were then analyzed by SDS-PAGE using NuPAGE® 4-12% Bis-Tris Gel, NuPAGE MES SDS running buffer (Invitrogen Carlsbad, CA) and visualized by Coomassie staining (Bio-Rad, Hercules, CA).
Restoration of complement classical pathway
The activity of purified rhC2 was determined using the Total Complement (3-pathway) ELISA (Alpco, Salem, NH) according to manufacturer instructions. Pooled human serum that had been stripped of C2 by affinity purification (EMD BioSciences, San Diego, CA) was reconstituted with various concentrations of rhC2 and tested. The functional activity of rhC2 was also analyzed by hemolysis in gel assays that detect deficiencies in the classical and alternative pathways of complement activation as well as deficiencies in the terminal sequence. In the classical pathway assay antibody-coated sheep red blood cells were suspended in 0.6% agarose (SeaKemR ME agarose, Cambrex BioScience Rockland Inc., Rockland, ME) in the presence of Ca2+ and Mg2+. Guinea pig erythrocytes in the presence of Mg2+ and EGTA were used similarly in the assay for the alternative pathway. Samples were added to holes punched in the agarose in 5 μl volumes and allowed to diffuse into the agar in moist atmosphere at 4°C for 18 hours and then at 37°C for 2 hours for the lytic reaction to proceed .
Deposition of C3 fragments on bacteria
Streptococcus pneumoniae 23F, obtained from Statens Serum Institut (Copenhagen, Denmark) were cultured on blood agar plates and then grown in Todd-Hewitt broth until log-phase was reached. The bacteria were harvested, washed in 0.15 M NaCl and killed by adding 0.8% glutaraldehyde in 0.15 M NaCl for 20 min. The killed bacteria were stored in small aliquots at -80°C until use. To measure C3 deposition on the bacteria, serum (100 μl), diluted 1/20 in VBSCaMg, was added to 2.5 μl bacteria and incubated for 30 min at 37°C with continuous agitation. The incubation was stopped by adding 500 μl of ice-cold VBSCaMg. The bacteria were washed (centrifugation 2,880 × g, 15 min) and then 100 μl of anti-C3d (Quidel, Santa Clara, CA), diluted 1/100 in VBSCaMg was added followed by an incubation at 4°C for 30 min. After the incubation the bacteria were washed and resuspended in 100 μl VBSCaMg and 5 μl FITC-conjugated polyclonal F(ab')2 rabbit anti-mouse immunoglobulins (DakoCytomation, Glostrup, Denmark) was added. The tubes were then incubated for 30 min at 4°C. Prior to analysis by flow cytometry (Epics XL-MCL Beckman-Coulter, Fullerton, CA), the bacteria were washed once and resuspended in 500 μl VBSCaMg.
Sera from 6 patients with homozygous C2 deficiency were used. Studies on the C2-deficient patients were approved by the Lund University Research Ethics Committee (LU 513-01) and written informed consent was obtained for each patient. The 6 individuals all had type I C2 deficiency as ascertained by DNA analysis, and had been reported earlier . MBL genotypes had also been determined as reported . Only one of these patients was homozygous for the wild-type variant of the structural MBL2 gene giving rise to high concentration of MBL and lectin pathway activity. The others were heterozygous for this allele and depending on promoter variants they showed lower concentrations of serum MBL to varying degrees.