A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates

Preparation of the immunogen

Human serum diluted in PBS was injected into a size exclusion HPLC column (Ultraspherogel SEC 4000; Beckman Instruments, San Ramon, CA, USA) using PBS as mobile phase. The peak eluting at 5.16 min representing the void volume of the column was collected into four fractions (Fig. S1). These fractions contained the C3 protein and C3-associates together with other large serum proteins. These protein moieties contained in these fractions were pooled and concentrated using Centricon-30 (Amicon Co., Beverly, MA, USA) and further purified by re-chromatography.

Antibody generation

A 4 week old Balb C mouse was immunized with human complement factor C3 (60 µg) purified from human serum by size exclusion chromatography as described above. Following three immunizations in a 2 weeks interval spleen cells were fuzed with the hypoxanthine–aminopterin–thymidine sensitive murine myeloma cell line 63A8.536 using the poly ethylene glycol method as described earlier (Wagner et al., 1996). Outgrowing clones were screened by a multi-slot immunoblotting device (Millipore, Bedford, MA, USA) (Fig. S2). Clones recognizing SDS denatured C3 under non-reducing conditions were kept, sub-cloned and further used for experiments. Clone number 18 as depicted in Fig. S2 was selected for its capability in immune purification of the C3 molecule. This antibody was finally named 3F7E2.

Purification of 3F7E2-antibody

The well producing antibody clone 3F7E2 was grown for 72 h in tissue culture flasks using RPMI1640 supplemented with 10% fetal calf serum containing penicillin/streptomycin as culture medium. The incubation conditions at a humidified (95% H₂O) incubator were 5% CO₂ and 37 °C. The tissue culture supernatant was filtered through a 0.22 µm filter. The resultant tissue culture fluid was loaded onto a HiTrap protein G column (GE Healthcare, Uppsala, Sweden) for purification of the murine IgG1 mAb. The purified mAb was then dialyzed against phosphate-buffered saline (PBS) at 4 °C and then further used for preparing an immunoaffinity column.

Gel electrophoresis and immunoblotting

One µL of human serum was diluted in near neutral pH sample buffer and loaded onto a PAGE gel (10%) and run using a Tris Glycine buffer omitting SDS for native gel and including SDS for C3a/C3 protein characterization. For detection of C3a 12% Mini-Protean TGX™ gels (456-1046; BioRad, Munich, Germany) and a Tris-Tricine SDS running buffer (161-0744; BioRad, Munich, Germany) were used. Native C3a was obtained from Complement Technology Inc. (Tyler, TX, USA), which had been purified from human donors, whereby its natural structure had been preserved.

Gels were blotted onto nitrocellulose using a semidry blotting device. The blotted nitrocellulose membrane was blocked with BM Chemiluminescence Blotting Substrate (POD) (Roche Molecular Biochemicals, Mannheim, Germany) and then developed using 3F7E2-mAb by incubating over night at 4 °C. Following two washes in Tween–phosphate buffered saline (TPBS) the blot was further incubated with POX conjugated goat anti mouse Ab 1:10,000 (PO447, DAKO, Denmark) for 1 h at room temperature and further developed using the chemiluminescence method with BM Chemiluminescence Blotting Substrate (POD) (Roche Molecular Biochemicals, Mannheim, Germany) and recording the site of antibody binding with luminescence imaging. Images were recorded using Fusion 3 software at the image analyzer unit (Fusion FX, Vilber Lourmat, Eberhardzell, Germany). C3 activation experiments were carried out by incubation of serum at 37 °C for various time periods. Obtained C3 split products were identified by immunoblotting.

Functional study using Zymosan activation

Ten µL of human serum were incubated in polypropylene reaction tubes of 0.5 mL (Sarstedt, Nuembrecht, Germany) with 5 µg and 10 µg of activated Zymosan for 15 min at 37 °C. As controls the antibody alone or zymosan suspension buffer alone were included in the test. The activation process was stopped by adding 1 µL of the experimental volume into sample buffer containing EDTA, which was heated for 3 min at 95 °C and loaded onto an SDS Page Gel (12% Mini-Protean TGX™ gel, 456–1046; BioRad, Munich, Germany) under nonreducing conditions. The 12% gel was run using Tris-Tricine SDS buffer (BioRad, Munich, Germany) and the resultant gel was transferred to nitrocellulose by semidry blotting and further developed using either mAb 3F7E2 or ab48342, rabbit polyclonal antibody to C3 (Abcam, Cambridge, UK) as described above.

C3 ELISA

Goat anti mouse precoated plates (Reacti-Bind™ Antibody Coated Plates, Pierce Biotechnology, Rockford, IL, USA) were washed once with PBS and the 3F7E2-mAb was bound onto the plate by incubating hybridoma cell supernatant onto the plate for 2 h. After one wash with TPBS 100 µL of samples were loaded together with a C3 standard series. Following an incubation period of 2 h the ELISA plate was washed three times with TPBS using an ELISA washing machine and the rabbit anti human C3 Ab conjugated with biotin (ab48342; Abcam, Cambridge, UK) diluted 1:2,500 using Assay Diluent (EL-ITEME2, RayBiotech Inc., Norcross, GA, USA) was incubated for 1.5 h under constant shaking. Following three washes with the ELISA washing machine with TPBS the streptavidin HRP was bound to the biotin while incubating in Assay Diluent (EL-ITEME2, RayBiotech Inc., Norcross, GA, USA) for 30 min at room temperature under constant shaking. Following three washes with TPBS the TMB substrate chromogen solution (Peroxidase Substrate Solution, KPL, Gaithersburg, MD, USA) was added and kept in dark for 10 min. The reaction was stopped with 1 M HCl and read with the ELISA reader (405 nm). Each value was calculated according the standard curve and samples were measured in quadruplicate.

Production of a 3F7E2-immunoaffinity column

Protein G column purified IgG 1 mAb was ligated to cyanogen bromide Sepharose for immunoaffinity purification of human factor C3a/C3. The 3F7E2-sepharose column was washed with wash buffer 1 (50 mM Tris-HCl, 0.5 M NaCl, 0.1% Triton X-100, pH 8.0) and wash buffer 2 (50 mM Tris-HCl, 0.5 M NaCl, 0.1% Triton X-100, pH 9.0) and finally with triethanolamine (TEA) elution buffer (50 mM TEA, pH 11.3). Following the preparation procedure of the immunoaffinity column human precleared serum was loaded onto the 3F7E2-mAb column by recirculation at 4 °C for 2 h. The loaded column was then washed with 10 column volumes of wash buffer (0.01 M Tris-HC1, pH 8.0, at 4 °C, 0.14 M NaC1, 0.025% NaN₃, 0.5% Triton X-100 and 0.5% sodium deoxycholate), 10 column volumes of wash buffer 1 followed by wash buffer 2. Following the washing procedure, the protein was eluted from the 3F7E2-column using TEA elution buffer, pH 11.3 into 1 M Tris-HCl, pH 6.7. Purified C3a/C3 was loaded onto 10% SDS PAGE gels and stained with Coomassie Brilliant Blue followed by destaining. Destained gels were incubated in glycerol H₂O for 1 h. Individually stained bands were cut out and subjected to proteomic analysis.

A haptoglobin immunoaffinity column (3A8-mAb) was prepared using the same method as described earlier (Wagner et al., 1996). Protein affinity purification and electrophoresis was performed using the same method as described for C3 affinity purification.

Protein gel electrophoresis for proteomic analysis

Equal amount of protein originating from haptoglobin and C3 immunoaffinity purification, respectively, were loaded onto separate lanes and different SDS PAGE gels both run under reducing conditions. The gels were stained by Coomassie Brilliant Blue followed by destaining. From both the C3 and haptoglobin containing lanes the same sized slice was cut out covering the entire molecular weight range and submitted for proteomic analysis.

Mass spectrometry

Ethics approval and consent to participate

All experiments were performed in accordance to the Helsinki Declaration and approved by the Ethics Committee of the Medical University of Vienna (EK721/2007). Serum samples were collected from adult subjects older than 18 years of age who gave written informed consent. All methods have been performed in accordance with the current regulations and guidelines. Animals were kept at the Center for Biomedical Research at the Medical University of Vienna. The Austrian Federal Ministry of Education, Science and Research provided full approval for this research (BMWF66.009/0046-II) in accordance to the European Convention for the Protection of Vertebrate Animals used for scientific purposes.

Immunoblotting of C3a/C3 and antibody specificity

By testing the 3F7E2-mAb for its specific recognition site at the C3a/C3 molecule, it was demonstrated that there is no binding under reducing conditions (Fig. 1B). Immunoblotting recognized C3 at a molecular weight of 190 kDa and more specifically, commercial C3a obtained from Complement Technology Inc. (Tyler, TX, USA) at a molecular size of approx. 9 kDa when the PAGE gel was run under non-reducing conditions (Fig. 1A). A conformational structure together with a specific amino acid sequence located at the anterior part within the C3a molecule was revealed to represent the antibody binding site.

Native gel electrophoresis

Initial size exclusion chromatography experiments for preparing the immunogen (Fig. S1) led to the hypothesis that C3 is present in association with other proteins under native conditions. In order to analyze the hydrodynamic size of the molecule and verify that the native C3a/C3 protein is associated in human serum with further proteins we determined its mobility in native gel. This revealed a spreading of C3 positive bands over a broad range in each of nine tested individuals and the pattern was unique to each individual. Nevertheless, the main dominant band in all samples was at a size of 190 kDa, which complies with the expected size of native C3 (Fig. S3).

Functional analysis of 3F7E2-mAb

Functional tests were performed to show a possible biological activity of 3F7E2-mAb in the activation process of the complement cascade. The activation process of human serum was initiated with activated Zymosan at 37 °C. An inhibitory potential of more than 70% (assessed by densitometric scanning) was achieved when purified 3F7E2-mAb was added before addition of Zymosan and the incubation period was restricted to 15 min (Fig. 2). Similar findings could be obtained by ELISA testing after 15 min Zymosan activation, which showed an inhibition of C3 consumption by 70% via addition of 3F7E2-mAb (490.1 ng/mL ± 20.1 ng/mL vs. 150.3 ng/mL ± 9.7 ng/mL).

Immunoapheresis of C3a/C3 and identification of bands

In a further technique to define the complex molecular structure, we sought for a way of immunopurification of these C3a/C3 compounds. For this purpose we generated an immunoaffinity 3F7E2-chromatography column for immunoapheresis of native C3a/C3 from human serum. Following the selection of the mAb 3F7E2 and preparation of the column, the C3a and C3 moiety (Fig. 3) could be purified under native conditions. The eluted fractions using TEA buffer were loaded onto an SDS-PAGE gel and electrophoresed under non-reducing conditions.

After SDS PAGE gel electrophoresis of eluted C3 fractions, staining of the gel using Coomassie Brilliant Blue revealed the main C3 band at 190 kDa and C3a as well as a C3 degradation product band at 9 and 8 kDa, respectively (Fig. 4).

Proteomic identification of C3a/C3 associates

Narrowly cut sections of the 9 kDa and 8 kDa bands were submitted to proteomic analysis. The resultant peptide finger print revealed peptides matching to amino acids 672–748 complying with the primary structure of full length C3a. The peptide fingerprint of the marginally smaller band (8 kDa) was also matching with the C3a sequence however missing a fragment from C-terminal half (Table 1) and most likely represents a degradation product. The peptide finger print of the 190 kDa band provided full length C3 sequence peptides (Fig. 4).

In the proteomic analysis a number of proteins or their fragmentation products were co-purified. Out of 278 proteomically identified proteins, 39 fulfilled the cut off criteria for specifically binding to the C3a/C3 molecule obtained by immunoapheresis. This cut off criteria were defined as proteins with strong (>10-fold) and consistent (p-value < 0.05) regulation in three technical replicates, consistent with C3a/C3-specific interaction. As depicted in Fig. 5 these 39 proteins, also listed in Table 2, had an at least 10-fold higher quantity of binding to C3a/C3 as compared to the haptoglobin control column.

In addition to some ubiquitous proteins and well-known C3 interaction partners, some particularly interesting associated proteins that may represent potential therapeutic targets were identified. Among the detected proteins were several biologically important factors, such as: (1) regulators of the complement cascade: Complement factor H, Complement factor H-related proteins 1 and 2, Complement C1q, Complement C4-B and C4b-binding protein and Complement component C8; (2) serine protease inhibitors: Serpin A12, Inter-alpha-trypsin inhibitor and Protein AMBP as well as (3) intracellular proteins such as Histone H2AX.

Further potential C3-interacting partners, with proteins below selected cutoff criteria and enrichment value above 0, located in the right half of the volcano plot of Fig. 5 are listed in Table S1.