Purification and characterization of RHP (factor H) and study of its interactions with the first component of complement.

RHP has been purified from the plasma of both normal individuals and patients with rheumatoid arthritis (RA). RHP from both these sources was shown to be identical with Factor H by reaction with antisera and N-terminal amino acid sequence analysis. Factor H, from both normal and RA sera, inhibited the solubilization of immune precipitates but did not affect prevention of immune precipitation. Factor H was shown to inhibit the haemolytic activity of fluid-phase C1, but unlike C1-inhibitor, it had little effect on C1 bound to EA (EAC1). Factor H was shown to complex with intact C1, to isolated C1q and to the C1r:C1s tetramer. However, binding of factor H to C1 did not dissociate the C1 macromolecule. A C1-Factor H complex was detected in the serum and plasma from normal individuals and patients with systemic lupus erythematosus and RA. Serum levels of this complex were reduced, by EDTA-treatment of serum and by activation of complement by the classical pathway.

Regulatory system of guinea-pig complement C3b: tests for compatibility of guinea-pig factors H and I with human factors.

Two proteins that are involved in cleavage of methylamine-treated C3 of guinea-pig origin (C3(MA)gp) have been isolated from guinea-pig serum. One of them functioned as a cofactor of human factor I (Ihu) for cleavage of C3(MA)gp and its molecular size was 150 kDa. The other was functionally pure and able to cleave C3(MA)gp together with human factor H (Hhu). They appear to be analogous to human factors H and I in the guinea-pig and will be referred to as Hgp and Igp. Methylamine-treated human C3 [C3(MA)hu] was not a compatible substrate for Hgp or Igp: little cleavage of C3(MA)hu was observed if human factor H (Hhu) or I was substituted with the guinea-pig counterpart. C3(MA)gp, on the other hand, served as a substrate, though less efficiently, for Hhu and Ihu. Human C4b-binding protein (C4bp) and membrane cofactor protein (MCP) as well as Hhu could participate in cleavage of C3(MA)gp by Igp or Ihu. In these assays, C3(MA)gp was degraded again less efficiently than C3(MA)hu. Interestingly, human C3b/C4b receptor (CR1) mediated factor I-dependent cleavage of C3(MA)hu and C3(MA)gp to a similar extent regardless the sources of factor I. These results suggest that factor I-dependent C3b regulatory system is species-specific except in the case of CR1, which may function as a cofactor irrespective of species.

Demonstration of the complement regulating protein, beta 1H, in skin biopsies from patients with bullous pemphigoid.

beta 1H-globulin is a recently characterized plasma protein which regulates the biologic activities of the major fragment of the third complement component, C3b. The major function of this protein is to act as a co-factor for C3b Inactivator (C3bINA) in the cleavage of C3b to an intermediate molecule, C3b', consisting of an intact beta-chain covalently bound by disulfide bridges to 2 alpha-chain fragments of 40,000 and 67,000 daltons. Final cleavage of C3b' to the C3c and C3d fragments requires an additional protease such as plasmin or elastase. Additionally, beta 1H interferes with the activity of the alternative pathway convertases, C3bBb and C3bBbP, by displacing or competing with the binding of factor B. In this study, perilesional skin biopsies from 10 patients with active bullous pemphigoid were examined for the presence of beta 1H at the dermal-epidermal junction by immunofluorescent methods. The protein was found in 8 of 9 biopsies in which C3 also was deposited. In a single case where C3 was not found, beta 1H was not seen. These findings suggest that beta 1H plays a role in the in vivo control of C3b and provides additional evidence for the participation of the complement system in the pathogenesis of bullous pemphigoid.

Isolation of human complement factors C3, C5 and H.

An improved method for simultaneous purification of complement factors C3, C5 and H from human plasma has been developed. Using an initial batch separation technique with QAE-Sephadex, followed by chromatography on SP-Sephadex and gel filtration in Sephadex G-200, 600 mg of highly pure C3 can be prepared from 1600 ml of plasma. Simultaneously about 70 mg of highly pure factor H and 30 mg of C5 are obtained by chromatography of post SP-Sephadex material on DEAE-Sephacel. A small amount of C3 in the C5 pool is removed by anti-C3-Sepharose. By maleylation or citraconylation of reduced and alkylated C3, the constitutive polypeptide chains are modified in a way that made them separable by ion exchange chromatography.

High resolution isoelectric focusing of immunoprecipitated proteins under denaturing conditions. A simple analytical method applied to the study of complement component polymorphisms.

A simple analytical method for the study of structural protein polymorphisms is described. It consists of the immunoprecipitation of non-radiolabeled proteins using monospecific polyclonal antibodies followed by isoelectric focusing (IEF) under completely denaturing conditions in vertical polyacrylamide slab gels. The method uses small amounts of sample (usually unfractionated plasma or serum), requires no sophisticated equipment and allows the screening of large numbers of samples with comparatively small effort. This method has been applied in the identification of 2 human complement-component polymorphisms, C4-binding protein (C4-bp) and factor H (beta 1H).

Isolation and characterization of rabbit H of the alternative complement pathway.

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.

Partial characterization of human complement factor H by protein and cDNA sequencing: homology with other complement and non-complement proteins.

Factor H, a control protein of the human complement system, is closely related in functional activity to two other complement control proteins, C4b-binding protein (C4bp) and complement receptor type 1 (CR1). C4bp is known to have an unusual primary structure consisting of eight homologous units each about 60 amino acids long. Such units also occur in the N-terminal regions of the complement proteins C2 and factor B, and in the non-complement serum glycoprotein beta 2I. Amino acid sequencing, and sequencing of a factor H cDNA clone, show that factor H also contains internal repeating units, and is homologous to the proteins listed above.

Isolation of bovine complement factor H.

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.

Human complement factor H: an additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement.

In addition to the 150-kDa factor H protein, we have previously described a 43-kDa factor H molecule in human plasma, which probably represents a translational product of the additional 1.8-kb mRNA for factor H. This factor H was isolated from human plasma by means of immunoaffinity chromatography and high-performance liquid chromatography. When tested for its functional activity, this purified 43-kDa H protein was shown to act as cofactor for factor I- mediated cleavage of fluid-phase C3b to iC3b.

Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography.

Human complement factor H was prepared in highly purified form from fresh serum by euglobulin precipitation, DEAE-Sephacel chromatography and Sephacryl S-300 gel filtration. This preparation allowed the recovery of 37% of the initial factor H. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that factor H was homogeneous both in reduced and non-reduced media and exhibited a molecular mass of 150 kDa. Charge-shift experiments clearly showed the presence of hydrophobic sites in the factor H molecule. Charge shifts were observed with two detergent systems (Triton/sodium deoxycholate and Triton/cetyltrimethylammonium bromide). Factor H was able to bind to phenyl-Sepharose. This property allowed us to study two populations of factor H. These two populations exhibited the same physicochemical parameters, but revealed differences in their ability to aggregate in low- and iso-ionic-strength media. The molecular basis and biological significance of this heterogeneity are discussed.

Purification of the human complement control protein C3b inactivator.

An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that beta 1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.

Simplified method for purification of mouse beta 1H.

A simple three-step method was described for purification of murine beta 1H, one of the essential regulatory proteins of complement system. The method consists of heparin-Sepharose affinity chromatography; gel filtration on a Sepharose 6B column, and DNA-cellulose affinity chromatography. By this method over 10 mg of beta 1H can be purified by more than 200-fold from 100-ml of EDTA serum of various strains. Overall yield of beta 1H was about 45%. The purified beta 1H was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. The purified mouse beta 1H showed physicochemical properties very similar to those described for human beta 1H: mouse beta 1H is a beta-globulin consisting of a single polypeptide chain of molecular weight of 160,000. Purified mouse beta 1H retained its functional activity as the essential cofactor for the cleavage of fluid-phase human C3b by the human C3b inactivator. Immunization of rabbits with the purified mouse beta 1H resulted in the production of the potent and monospecific antibody.

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography.

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.

Purification and structural studies on the complement-system control protein beta 1H (Factor H).

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.