Human Cord Blood Serum-Derived APP α-Secretase Cleavage Activity is Mediated by C1 Complement

Reagents and Antibodies

CBS was obtained from Lee Biosolutions (St. Louis, MO, USA). CBS was separated from umbilical cord blood by allowing clotting for 5–10 h in tubes without anticoagulant followed by centrifugation at 3500 rpm for 5–10 min and filtration through a membrane with a pore size of 0.22 µm. Individual CBS samples were prepared from a single lot of cord blood, and more than 10 samples of CBS were combined as “pooled CBS.” Normal human aged blood serum (AgBS, >75 years old) were obtained from Florida Blood Services (Tampa, FL, USA). All sera were heat-activated. Heat-inactivation of human sera was conducted at 56°C for 30 min. Complement proteins, complement depleted sera, and complement inhibitors were acquired from the following sources. Human complement protein C1 inhibitor (Cat# GF178), complement protein C1 from human serum (Cat# 204873), complement protein C3-depleted human serum (Cat# 234403), and complement protein C1q-depleted human sera (Cat# 234401) from MilliporeSigma (St. Louis, MO, USA). Complement protein C3 from human serum (Cat# A113) and complement iC3b from human serum (Cat# A115) from Complement Technology, Inc. (Tyler, Texas, USA). Complement protein C1q from human serum (Cat# 204876) from MilliporeSigma (St. Louis, MO, USA). Compstatin, complement protein C3b inhibitor human (Cat# SB 431542) was acquired from Tocris Bioscience (Minneapolis, MN, USA). Sera from C1qa deficient (def) mice (129P2/OlaHsd, 3 months of age) were kindly provided by Dr. Andrea J. Tenner (University of California, Irvine, USA). Antibodies used in this studies include: specific anti-sAPPα monoclonal antibody (2B3; IBL, Minneapolis, MN, USA, Cat# 11088 RRID: AB_494690), anti-APP C-terminal polyclonal antibody (pAb751/770; EMD Millipore, La Jolla, CA, USA, Cat# 171610, RRID: AB_211444), anti-N-terminal Aβ monoclonal antibody (6E10; Covance Research Products, Emeryville, CA, USA), and anti-β-actin monoclonal antibody (Cat# A5316, RRID: AB_476743) from MilliporeSigma (St. Louis, MO, USA). The human sAPPα ELISA assay kit was purchased from IBL AMERICA, Minneapolis, MN, USA.

CBS and AgBS Fractionation

In order to purify and characterize the α-secretase in CBS or AgBS, the Econo-Pack Serum IgG purification kit, and 10 DG columns (Bio-Rad, Philadelphia, PA, USA) were initially employed to remove highly abundant IgG and salts. The desalted serum was applied to DEAE Affi-Gel blue columns and residual IgG was eluted according to the instructions. Then, 20 additional protein fractions were collected by eluting with an ionic strength gradient of NaCl buffer ranging from 0.1 M to 2.0 M. The remaining proteins on the column were eluted by the regeneration buffer included in the kit and collected as the regeneration fraction. The 0.8 M NaCl eluted protein fractions were combined and sent to Moffitt Cancer Center protein purification core for further separation by size-exclusion chromatography, employing analytic Superdex 200 columns and eluting with phosphate-buffered saline (PBS), and ion-exchange chromatography, employing Q-Sepharose columns and eluting with 500 mM NaCl, 50 mM Tris, pH 7.6. The final enzyme containing fractions were exchanged to PBS buffer by Ultracel-10 membranes (10 kDa, Merck Millipore, Burlington, MA, USA) for further experimentation and referred to as α-CBSF or AgBSF (Fig. 1).

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Fig. 1.

Tentative workflow for α-CBSF and AgBSF fractionation. Initially, CBS and AgBS were desalted followed by sequential chromatographic separation using DEAE column, size-exclusion, and ion-exchange chromatography. Each fraction was collected followed by efficacy testing by treating CHO/APPwt cells for sAPPα release. Eventually a fraction (α-CBSF) was chosen based on the highest capacity for sAPPα generation. LC-MS/MS was performed to identify the source of the protein function. AgBS, aged blood serum; CBS, cord blood serum; DEAE, diethylaminoethyl; LC-MS/MS, liquid chromatography with tandem mass spectrometry; sAPPα, secreted amyloid precursor protein-α.

LC-MS/MS

Briefly, each lane gel slices were destained using acetonitrile and subjected to tris 2-carboxyethy-phosphine (TCEP) reduction and iodoacetamide alkylation followed by in-gel trypsin digestion, extraction, and vacuum centrifugation concentration. A nanoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA) coupled to an electrospray ion trap mass spectrometer (LTQ-Orbitrap, Thermo, San Jose, CA) was used for tandem mass spectrometry peptide sequencing experiments. A 5 µl sample was first loaded onto a pre-column (5 mm × 300 µm ID packed with C18 reversed-phase resin, 5 µm, 100 Å) and washed for 8 min with aqueous 2% acetonitrile and 0.04% trifluoroacetic acid. Next, the trapped peptides were eluted onto the analytical column (C18, 75 µm ID × 15 cm; Pepmap 100, Dionex, Sunnyvale, CA). The 120-min gradient was programmed as: 95% solvent A (2% acetonitrile + 0.1% formic acid) for 8 min; solvent B (90% acetonitrile + 0.1% formic acid) from 5% to 50% in 90 min; then solvent B from 50% to 90% in 7 min and held at 90% for 5 min; followed by solvent B from 90% to 5% in 1 min and re-equilibrate for 10 min (60-min gradient setup). The flow rate on the analytical column was 300 nl/min. Five tandem mass spectra were collected in a data-dependent fashion following each survey scan. The MS scans were performed in Orbitrap to obtain accurate peptide mass measurement and the MS/MS scans were performed in a linear ion trap using 60 s exclusion for previously sampled peptide peaks. The LC-MS/MS data were submitted to a local MASCOT server Swiss-Prot Human database for MS/MS protein identification search via ProteomeDiscoverer software. Two trypsin missed cleavages were allowed; the precursor mass tolerance was 1.08 Da. The MS/MS mass tolerance was 0.8 Da. The incorrect detection rate in each LC-MS/MS analysis was set to be less than 1%.

Cell Culture

Chinese hamster ovary (CHO) cell line with stable expression of human wild-type APP (CHO/APPwt) was a generous gift from Drs. Stefanie Hahn and Sascha Weggen (University of Heinrich Heine, Düsseldorf, Germany). These cells were initially genotyped to confirm their genetic make-up. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 100 U/ml of penicillin/streptomycin. For treatment, the cells were plated in a 24-well plate at 2 × 10 ⁵ cells/well for overnight incubation, washed, and treated with CBS, heat-inactivated (56°C, 30 min) CBS, AgBS, or α-CBSF, AgBSF in DMEM. After treatment, supernatants were collected and the cells were washed with ice-cold PBS three times and lysed with cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO4, 1 µg/ml leupeptin, and 1 mM PMSF; Cell Signaling Technology, Danvers, MA, USA). Both cell supernatants and lysates were used for sAPPα analysis by ELISA. In addition, murine N2a cells transfected with APPwt (N2a/APPwt) and primary neuronal cells were obtained from cerebral cortices of TgAPPwt mouse embryos, between 15 and 17 days in utero, as described previously ²⁶ . For TgAPPwt mouse-derived cortical neurons, cerebral cortices were isolated from TgAPPwt mice between 15 and 17 days in utero and mechanically dissociated in trypsin (0.25%) individually after incubation for 15 min at 37°C. Cells were collected after centrifugation at 1200 g, suspended in DMEM supplemented with 10% fetal calf serum, 10% horse serum, uridine (33.6 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), and fluorodeoxyuridine (13.6 mg/ml, Sigma-Aldrich), and seeded in 24-well collagen-coated culture plates at 2.5 × 10 ⁵ cells/well. After reaching confluence (∼70–80%) these cells were treated with purified human complement C1 at 0, 5, 10, or 20 µg/ml for 4 h, supernatants were collected for sAPPα measurement by ELISA and cells were washed with ice-cold PBS three times and lysed with cell lysis buffer followed by western blotting using 6E10 and pAb751/770 antibodies.

Transgenic APPwt Mice

Transgenic wild-type B6.Cg-Tg (PDGFB-APP) 5Lms/J strain (TgAPPwt) male and female mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in the Animal Facility in the College of Medicine at the University of South Florida and maintained on a 12 h light/12 h dark cycle at ambient temperature and humidity with ad libitum access to food and water. All animal experiments involving mice were performed in compliance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and in accordance with the guidelines of the University of South Florida Institutional Animal Care and Use Committee (IACUC reference number: IS00000438).

Western Blot Analysis and ELISA

Western blot (WB) analyses and quantification were performed as previously described ¹¹ . Briefly, the proteins from the cell-free suspensions, cell lysates, and homogenized tissue were electrophoretically separated using 10% bicine/tris gel (8 M urea) for proteins less than 5 kDa or 10% tris/SDS gels for larger proteins. Electrophoresed proteins were transferred to nitrocellulose membranes (Bio-Rad), washed and blocked for 1 h at room temperature in tris-buffered saline containing 5% (w/v) nonfat dry milk (TBS/NFDM). After blocking, membranes were hybridized overnight with various primary antibodies, washed, and incubated for 1 h with the appropriate HRP-conjugated secondary antibody in TBS/NFDM and developed using the luminol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Soluble sAPPα in the media was quantified with the IBL human sAPPα colorimetric sandwich ELISA kit according to the manufacturer’s instructions.

Statistical Analysis

Comparison between two groups were performed by Student’s t test analysis. For more than two groups, one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc analysis was used to compare each other for statistical significance. Alpha was set at 0.05 for all analyses. All molecular analyses were repeated three times in parallel. Data are expressed as mean ± SEM. The statistical package for the social sciences release IBM SPSS 23.0 (IBM, Armonk, NY, USA) was used for all data analyses.

Heat-Sensitive APP α-Secretase Activity in CBS

In order to identify the source of α-secretase in CBS, we initially confirmed that CBS possesses heat-labile α-secretase activity. CHO/APPwt cells were treated with CBS (2%) for 0, 5, 10, 15, 30, 60, and 120 min, followed by determination of sAPPα in conditioned medium by ELISA. CBS caused a time-dependent increase in sAPPα release from CHO/APPwt cells that was significantly greater than the control treatment (Fig. 2a; *P < 0.05, **P < 0.01, ***P < 0.001). Similarly, CHO/APPwt cells were treated for 2 h with 0, 0.25, 0.5, 1, 2, and 5% CBS, followed by determination of sAPPα in conditioned medium by ELISA. CBS caused dose-dependent release of secreted amyloid precursor protein-α (sAPPα) in culture medium from CHO/APPwt cells that was significantly greater than the control treatment (Fig. 2b; *P < 0.05, **P < 0.01, ***P < 0.001). Next, we wanted to determine whether heat-inactivation of serum reduces the sAPPα-producing capacity of CBS. CHO/APPwt cells were treated with 2% whole or heat-inactivated CBS or AgBS (56°C for 30 min) for 2 h followed by determination of sAPPα in conditioned media by ELISA. Both CBS and AgBS heat-inactivation significantly limited sAPPα-releasing capacity (Fig. 2c; *P < 0.05, **P < 0.01, ***P < 0.001). Furthermore, CHO/APPwt cells were treated with 2% whole or heat-inactivated α-CBSF or AgBSF for 2 h followed by determination of sAPPα in conditioned media by ELISA (Fig. 2d; *P < 0.05, **P < 0.01, ***P < 0.001). As shown most recently, purification significantly enhanced the sAPPα-producing capacity of CBS and AgBS, which again was significantly limited by heat-inactivation. These results confirm that CBS possesses an enhanced level of heat-sensitive α-secretase activity.

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Fig. 2.

CBS significantly enhances APP α-cleavage in CHO/APPwt cells. (a) CHO cells overexpressing human wild-type APP (CHO/APPwt cells) were treated for 0–120 min with CBS (2%). (b) CHO/APPwt cells were treated for 2 h with 0, 0.25, 0.5, 1, 2, and 5% CBS. (c) CHO/APPwt cells were treated for 2 h with PBS (0% serum, Ctrl), heat-inactivated CBS (Heat CBS), heat-inactivated AgBS (Heat AgBS), AgBS, or CBS at 2%. (d) CHO/APPwt cells were treated for 2 h with PBS (0% serum, Ctrl), heat-inactivated α-CBSF (Heat αCBSF), heat-inactivated AgBSF (Heat AgBSF), AgBSF, or α-CBSF at 2%, each purified as briefly described under “Materials and Methods.” Release of sAPPα into the conditioned medium after treatment was analyzed by ELISA. Data are presented as mean (±SEM) of sAPPα produced (ng/ml or ng/mg) from three independent experiments performed in triplicate. Significant differences from control treatment are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). AgBSF, aged blood serum fraction; CBS, cord blood serum; PBS, phosphate-buffered saline; sAPPα, secreted amyloid precursor protein-α; SEM, standard error of the mean.

Proteomic Analyses of CBSF and AgBSF

Having shown that CBSF possesses enhanced α-secretase activity, we next set out to determine the molecular source of α-secretase in CBSF and AgBSF by LC-MS/MS analysis. From 142 major proteins identified, we selected those most likely to exhibit α-secretase activity. Several of the major proteins identified in α-CBSF and AgBSF are shown in Fig. 3. Of these proteins, the subunits of complement protein C1 complex (C1q, C1r, and C1s) and alpha-2-macroglobulin showed significantly greater levels in α-CBSF compared with AgBSF (Fig. 3; *P < 0.05, **P < 0.01). Notably, the levels of complement proteins C3, C4, C5, C6, and C8 complexes, ApoE, ApoB100, and full-length APP (fAPP) were not significantly different between α-CBSF and AgBSF (P > 0.05). Complement proteins C1 and C3b complex have proteolytic activity, which can be inactivated at 56°C for 30 min. These proteins play important roles in neurodevelopment and provide early protection against stress or disease, including AD. Thus, we hypothesized that the heat-labile α-secretase activity in CBS is mediated in part by either complement protein C1 complex or C3b.

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Fig. 3.

Identification of proteomic profile of α-CBSF and AgBSF. The proteome of α-CBSF and AgBSF were analyzed by LC-MS/MS. From 142 major proteins identified, those exhibiting proteolytic and therefore potential α-secretase-like activity were selected. Of these proteins, the subunits of complement protein C1q (**P < 0.01), C1r (*P < 0.05), and C1s (*P < 0.05), and alpha-2-macroglobulin (*P < 0.05) showed significantly greater levels in α-CBSF compared with AgBSF. Data are presented as mean (±SEM) of number of peptide hits from three independent experiments. Significant difference between AgBSF and α-CBSF indicated by asterisk (*P < 0.05, **P < 0.001, ***P < 0.01). AgBSF, aged blood serum fraction; LC-MS/MS, liquid chromatography with tandem mass spectrometry; SEM, standard error of the mean.

CBS-Mediated α-Secretase-Like Cleavage of APP is Independent of Complement C3b

In order to determine whether α-secretase activity of CBS is mediated by activation of complement protein fragment C3b, we investigated whether depletion of complement protein C3 could limit the α-secretase-like activity of CBS. CHO/APPwt cells were treated with C3-depleted CBS (2%) for 0, 15, 30, 60, and 120 min, followed by determination of sAPPα in conditioned medium by ELISA. Depletion of C3 complement protein did not change sAPPα-producing capacity of CBS in CHO/APPwt cells (Fig. 4d; *P < 0.05, **P < 0.01, ***P < 0.001). In order to confirm that C3b complement does not mediate α-secretase activity in CBS, we preincubated whole or C3-depleted CBS (2%) with the C3b inhibitor compstatin at 0, 6, 12, 25, 50, and 100 µM for 15 min at 37°C, and then treated CHO/APPwt cells with these premix complexes separately for 2 h. Compstatin did not alter sAPPα production significantly elicited by whole (Fig. 4a; P > 0.05) or C3-depleted serum (Fig. 4b; P > 0.05). Therefore, CBS α-secretase-like activity is independent of enzymatic activity of complement C3b protein.

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Fig. 4.

CBS-mediated sAPPα release by CHO/APPwt cells is independent of complement protein C3b. C3b inhibitor compstatin at 0, 6, 12, 25, 50, and 100 µM was preincubated with CBS (2%) as well as C3-depleted CBS (2%) for 15 min. (a). CHO/APPwt cells were treated for 2 h with CBS (2%) + compstatin. (b) CHO/APPwt cells were treated for 2 h with C3-depleted CBS (2%) + compstatin. (c) In addition, CBS (2%) was preincubated with iC3b at indicated concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml). CHO/APPwt cells were treated for 2 h with CBS + iC3b. (d). CHO/APPwt cells were treated with C3-depleted CBS (2%) for 0, 15, 30, 60, and 120 min. Release of sAPPα into the conditioned medium after treatment was analyzed by ELISA. Data are presented as mean (±SEM) of sAPPα produced (ng/mg) from three independent experiments performed in triplicate. Significant differences from control treatment indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001). CBS, cord blood serum; ELISA, enzyme-linked immunosorbent assay; sAPPα, secreted amyloid precursor protein-α; SEM, standard error of the mean.

In order to determine whether complement protein component C3b acts as an inhibitor of CBS α-secretase-like activity, CHO/APPwt cells were treated for 2 h with premixes of CBS (2%) and iC3b (0, 1.25, 2.5, 5, 10, and 20 µg/ml), the proteolytically inactive product of C3b that prevents complement cascade activation. The sAPPα-producing capacity of CBS was not altered by iC3b as determined by sAPPα ELISA (Fig. 4c; P > 0.05), indicating that complement C3b protein does not act as a potent CBS α-secretase inhibitor.

CBS α-Secretase-Like Activity is Mediated in Part by Complement Protein C1 Complex

To determine whether α-secretase-like activity in CBS is mediated by complement protein C1 complex, we determined CBS α-secretase activity in the presence of complement protein C1 inhibitor. CHO/APPwt cells were treated with α-CBSF (1 and 2%) in the presence of complement protein C1 inhibitor at 0, 50, 100, and 200 µg/ml for 2 h. C1 inhibitor markedly reduced sAPPα released by α-CBSF (1 and 2%) in a dose-dependent fashion, as determined by ELISA (Fig. 5a; *P < 0.05, **P < 0.01, ***P < 0.001) and WB (2% CBS; Fig. 5b; *P < 0.05, **P < 0.01, ***P < 0.001; quantification not shown), indicating that α-secretase-like activity in CBS is mediated in part by complement C1 protein. In addition, we determined the effect of purified human C1 complement protein on sAPPα production in N2a/APPwt and TgAPPwt/primary neuronal cells in the absence of sera. Complement protein C1 dose-dependently increased sAPPα and α-CTF production in N2a/APPwt cells (Fig. 5c–e; **P < 0.01, ***P < 0.001) and TgAPPwt/primary neuronal cells (Fig. 5f–h; *P < 0.05, **P < 0.01) in the absence of serum, as determined by ELISA and WB. These results indicate that complement protein C1 complex mediates α-secretase-like activity in CBS.

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Fig. 5.

A specific complement protein C1 inhibitor significantly reduces APP α-cleavage induced by CBS. (a) CHO/APPwt cells were treated for 2 h with α-CBSF (1 and 2%) in the presence of C1 inhibitor at 0, 50, 100, and 200 μg/ml, followed by analysis of sAPPα in conditioned media by ELISA. (b) In addition, CHO/APPwt cells were treated for 2 h with α-CBSF (2%) in the presence of complement protein C1 inhibitor at 0–200 μg/ml, followed by analysis of sAPPα levels by WB. N2a/APPwt cells (c) and TgAPPwt/mouse primary neuronal cells (f) were treated with purified human complement protein C1 complex at 0, 5, 10, and 20 µg/ml in the serum-free condition for 4 h followed by analysis of holo APP, sAPPα and α-CTF in cell lysates by WB (c,f). The levels of sAPPα in conditioned media was also determined by ELISA (d,g) and band density radios of α-CTF to (full-length) holo APP are presented below each blot (e,h). Data presented as mean (± SEM) of sAPPα produced (ng/ml or ng/mg) from three independent experiments performed in triplicate. Significant differences from control treatment indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001). α-CTF, alpha carboxyl-terminal fragment; ELISA, enzyme-linked immunosorbent assay; sAPPα, secreted amyloid precursor protein-α; SEM, standard error of the mean; WB, western blot.

In order to confirm that complement C1 but not C3 protein has α-secretase-like activity, CHO/APPwt cells were treated with purified human complement C1 or C3, or C1q subunit, at 0, 2.5, 5, 10, and 20 µg/ml in serum-free media for 2 h followed by determination of sAPPα in conditioned medium by ELISA. As expected, complement protein C1 significantly increased sAPPα production in a dose-dependent fashion, whereas C1q alone minimally increased and complement protein C3 did not increase sAPPα production in serum-free medium (Fig. 6a; *P < 0.05, **P < 0.01). In contrast, both complement C1 and C1q protein component significantly increased sAPPα production in CHO/APPwt cells cultured in C1q-depleted sera (Fig. 6b; **P < 0.01, ***P < 0.001), and complement protein C1 significantly increased sAPPα and α-CTF level, while decreasing Aβ and β-CTF level in CHO/APPwt cells in the presence of whole sera as determined by WB (Fig. 6c).

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Fig. 6.

The purified human complement protein C1 complex promotes APP α-cleavage. (a) CHO/APPwt cells were treated for 2 h with purified human complement C1q subcomponent, C1 or C3, at 0, 2.5, 5, 10, and 20 µg/ml in serum-free media followed by analysis of sAPPα in conditioned media by ELISA. (b) CHO/APPwt cells were treated for 2 h with purified human complement C1q subcomponent, C1, and C3, at 0, 2.5, 5, 10, and 20 µg/ml in C1q-depleted serum followed by analysis of sAPPα in conditioned media by ELISA. (c) In order to further determine the effect of complement protein C1 on APP processing, CHO/APPwt cells were treated for 2 h with purified complement protein C1 at 0, 2.5, 5, 10, and 20 µg/ml in the absence of whole serum and cell lysates were prepared from each treated cell culture for analysis of holo APP, sAPPα, Aβ, and α- and β-CTF by WB. (d) CHO/APPwt cells were treated for 2 h with plasma (2%) obtained from C1qa-deficient mice or wild-type littermates at 3 months of age (n = 5 males or 3 females for each group) followed by determination of sAPPα in conditioned media by ELISA. For (a) and (b), data presented as mean (±SEM) of sAPPα produced (ng/ml) from three independent experiments performed in triplicate. Significant differences from control treatment indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001).

In order to investigate the role of complement protein C1 in mediating α-secretase activity in whole blood plasma, CHO/APPwt cells were treated with plasma from C1qa-deficient mice or age-matched wild-type littermates. Interestingly, the production of sAPPα elicited by plasma from male C1qa-deficient mice was not significantly different (Fig. 6d; P > 0.05), whereas that elicited by plasma from female C1qa-deficient mice was significantly greater than that elicited by age- and gender-matched plasma from wild-type littermates (Fig. 6d; **P < 0.01). Therefore, plasma α-secretase activity is not significantly altered by C1qa deficiency in male mice but significantly enhanced by C1qa deficiency in female mice. These results are consistent with C1-mediated α-secretase playing an important role in male and female individuals, but with other α-secretases compensating in its absence. In addition, the elevated sAPPα production capacity in female compared to male C1qa-deficient mice indicate an unknown function of this neurotrophic fragment in the peripheral system of females.

Altogether, these results confirm that APP α-secretase-like activity in human blood serum is mediated by complement protein C1 complex. The inability of C1q to rescue α-secretase activity in serum-free media is likely due to the absence of components C1s and C1r, which are required to complex with C1q in C1 for proteolytic activity.