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

Alzheimer's Disease (AD) is the leading cause of dementia in the elderly. In healthy individuals, amyloid precursor protein (APP) is cleaved by α-secretase, generating soluble α-amyloid precursor protein (sAPPα), which contributes neuroprotective functions in the neuronal environment. In contrast, in the neurodegenerative environment of AD patients, amyloid-ß-peptide (Aß) of either 40 or 42 residues are generated by increased activity of ß- and γ-secretase. These proteins amalgamate in specific regions of the brain, which disrupts neuronal functions and leads to cognitive impairment. Human umbilical cord blood cells (HUCBC) have proven useful as potential immunomodulatory therapies in various models of neurodegenerative diseases, including AD. Our most recent work studied the impact of umbilical cord blood serum (CBS) on modulation of sAPPα production. Heat-sensitive CBS significantly promoted sAPPα production, indicating that heat-sensitive factor(s) play(s) a role in this process. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to determine the molecular source of α-secretase in purified CBS and aged blood serum (AgBS) fraction. Of the proteins identified, the subunits of C1 complex (C1q, C1r, and C1s) and alpha-2-macroglobulin showed significantly greater levels in purified α-CBS fraction (α-CBSF) compared with the AgBS fraction (AgBSF). Specifically, C1 markedly increased sAPPα and alpha-carboxyl-terminal fragment (α-CTF) production in a dose-dependent fashion, whereas C1q alone only minimally increased and C3 did not increase sAPPα production in the absence of sera. Furthermore, C1q markedly increased sAPPα and α-CTF, while decreasing Aß, in CHO/APPwt cells cultured in the presence of whole sera. These results confirm our initial assumption that APP α-secretase activity in human blood serum is mediated by complement C1, opening a potential therapeutic modality for the future of AD.

A small fraction of dermatan sulfate with significantly increased anticoagulant activity was selected by interaction with the first complement protein.

Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1-->3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution.

The first component of the human complement system recognizes the active fraction of heparin.

A low molecular weight heparin fraction with high anticoagulant activity was isolated from the precipitate of the interaction with the first component of the human complement system. Our results confirmed the predictions: under very strict conditions of pH (6.3), CaCl2 concentration (2 mM), ionic strength (25 mM) and protein/heparin ratio (1/1), the first component of the complement recognizes the high antithrombin III affinity fraction of heparin, and allows a concentration of the biological activity of the original low molecular weight-heparin.

Identification of defensin binding to C1 complement.

In human serum we found strong defensin binding to the complexes of activated C1 complement (C1) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer (r2s2) and C1i did not bind defensin. When r2s2 was dissociated by EDTA, only the activated C1s (C1s) bound defensin. Binding of defensins to C1 complement represents a newly recognized bridge between the complement- and phagocyte-mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.

Rapid isolation and biochemical characterization of rat C1 and C1q.

Using a human IgG-Sepharose column to which rabbit anti-human IgG was bound (rabbit anti-human/human IgG-Sepharose), human and rat C1 or C1q were isolated from serum in a single step, and the C1q further purified to homogeneity by FPLC. This procedure allowed the rapid isolation of haemolytically active C1 or C1q, with a yield equal to or greater than published methods. The availability of human and rat C1q allowed comparison of the two molecules, revealing differences in their mobility on SDS-PAGE as well as on agarose gel electrophoresis. Amino terminal sequence analysis demonstrated greater than 78% residue identity between rat C1q A, B and C chains and the published human and mouse sequences. Similar amino acid compositions suggest that the homology extends throughout the molecules. In addition to the major A:B and C:C dimer bands, rat, unlike human C1q, contained minor dimer species. These may reflect heterogeneity in glycosylation and or lysine and proline hydroxylation.

Inorganic supports coated with N-substituted polyacrylamides: application to biospecific chromatography of proteins.

Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the gamma-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.

Purification of C8 and C9 from rat serum.

A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).

[Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent]. / Stupenchataia dissotsiatsiia subkomponentov C1, pervogo komponenta komplementa cheloveka, pri aktivatsii na affinnom sorbent.

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome.

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.

Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis.

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.

[A method of isolating C1q from human serum and its use in the solid-phase immunoenzyme determination of immune complexes]. / Metod vydeleniia C1q iz syvorotki cheloveka i ispol'zovaniia ego v tverdofaznom immunofermentnom opredelenii immunnykh kompleksov.

C1q was isolated from human serum by dialysis in 0.24 M EDTA, followed by affinity chromatography on immobilized IgG and removal of IgG traces in a column with anti-IgG antibodies. Microplates were coated with C1q in PBS at 10-20 mg/l, nonspecific binding sites were saturated with human serum albumin. The sera were diluted 16-fold in 0.05 M PBS, 0.01 M EDTA, 0.05% Tween. After incubation with diluted samples the plates were treated with horseradish peroxidase--anti-human IgG conjugates. Enzymic activity was measured by adding p-phenylenediamine (0.2 g/l) in acetate buffer, pH 5.9, containing 0.05% H2O2. The sensitivity of the assay ranged between 2.5 and 300 mg/l.

Further evidence for dilution-dependent dissociation of C1q in human serum.

Dissociation of the C1q subcomponent in native C1 upon dilution was reexamined by using ultracentrifugation in a sucrose gradient and high performance liquid chromatography system with a size exclusion column for separating the dissociated C1q fractions. The antigenic content of C1q in each fraction was detected by ELISA and Western blotting; binding to erythrocyte antibody was also determined. The results confirmed a previous claim that C1q in native C1 dissociated as a function of dilution: up to 14.5% of C1q antigen was in the low molecular weight form (approximate S value: 4-5). Commercial preparations of purified C1q also contained C1q antigen in the low molecular weight form.

A mechanism for the spontaneous activation of the first component of complement, C1, and its regulation by C1-inhibitor.

We have developed a method to initiate spontaneous activation of the first component of complement in serum, by the removal of C1-inhibitor through complexation with added C1s. Preliminary experiments to test this method using C1 reconstituted from its purified subcomponents led to an unexpected result: pre-incubation of the reassembled subcomponents with C1-inhibitor, followed by its removal with C1s, altered the subsequent pattern of spontaneous activation. Thus, pre-incubation with C1-inhibitor at 37 degrees C for 1 h resulted in sigmoidal activation of C1 with a prolonged lag phase. In contrast, pre-incubation with C1-inhibitor on ice for the same time resulted in subsequent rapid, pseudo first order activation of C1 with a half-life of about 5 min. We have examined the activation kinetics under a variety of conditions, and our data are consistent with a model proposed by Lepow and coworkers in 1965, involving both spontaneous activation and C1 catalyzed activation: (1) C1----k1 C1 (2) C1----k2C1 C1 According to this model, the role of C1-inhibitor is to eliminate the second step by rapidly forming a tight complex with C1 which becomes irreversible at 37 degrees C. When C1s was added to normal human serum, activation at 37 degrees C was also sigmoidal, similar to that of reconstituted C1.

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.

C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits.

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.

Purification and characterization of the 1q subcomponent of canine complement and its use in the 125I-C1q binding assay for detection of immune complexes.

The complement subcomponent, C1q, was isolated from serum obtained from clinically normal dogs, using a rapid 2-step process involving affinity chromatography. Yield of C1q ranged from 8 to 10 mg/L of serum. Hemolytically active C1q had 3 protein bands after sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and formed a single line of identity with rabbit anti-canine C1q. The amino acid composition of canine C1q was similar to that of human C1q and contained a high percentage of glycine. Isolated canine C1q was iodinated, and the fluid-phase binding assay was used to detect circulating immune complexes in dogs with systemic lupus erythematosus and rheumatoid arthritis.

Preliminary studies on the detection, isolation and partial characterization of Clq-containing IgM immune complexes in sera of patients with primary biliary cirrhosis.

The complement system is activated in primary biliary cirrhosis (PBC) and this activated state may be medicated by immunoreactive IgM. To identify and further characterize the relationship between the complement (Clq) and IgM in PBC sera, we developed an anti-Clq ELISA method which allowed detection of Clq-containing circulating immune-like complexes. Utilizing this technique, sera from 3 out of 5 patients with PBC revealed circulating immune-like complexes. Moreover, when serum samples were specifically examined for the presence of IgM containing Clq complexes, four of four samples examined were positive. Additional experiments indicated that these immune-like complexes could be removed from PBC sera by means of an anti-Clq immunoadsorbent. Upon subsequent isolation and characterization, these immune-like complexes demonstrated polypeptide chains corresponding to both human Clq and human IgM. Our experimental studies establish that Clq-containing IgM-like complexes can occur in the serum of patients with PBC, and provide additional support for the proposal that immunoreactive IgM can contribute to the activated complement system observed in PBC.

A rapid and efficient method for the purification of the complement subcomponents C1r and C1s in zymogen form using fast protein chromatography.

The purification of the subcomponents C1r and C1s of the first component of complement involves multiple steps and is time-consuming. This accounts for the frequently observed partial activation of the subcomponents. In this report we propose a simplified procedure of purification using a batch method and fast protein chromatography avoiding a shift of pH. The method provides C1r and C1s in a yield of 35 and 60% respectively. In addition, this study provides a simple and sensitive test to assess functional purity of C1r and C1s with respect to the other C1 subcomponents.

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.