Isolation and characterization of factor I of the bovine complement system.

OBJECTIVE: To isolate and characterize factor I of the bovine complement system. Sample Population-Serum obtained from the blood of beef cattle. PROCEDURES: Serum samples were fractionated to yield factor I by means of sequential precipitation, ion-exchange, and gel-filtration chromatography. The protein was identified throughout the procedure on the basis of its ability to degrade the alpha'-chain of bovine C3b in the presence of bovine factor H. Electrophoresis in polyacrylamide gels was used to assess the degradation of C3b and determine the molecular weights of factor I and its component polypeptide chains. RESULTS: Bovine factor I had an apparent molecular weight of 94 kd and consisted of 2 disulfide-bonded polypeptides that had apparent molecular weights of 51 and 42 kd (under reducing conditions). Factor H was required for the factor I cleavage of the alpha'-chain of bovine C3b into iC3b. A similar cofactor effect was provided by trypsinized bovine erythrocytes or erythrocyte ghosts. Bovine properdin was prepared and shown to be a single polypeptide chain of 58 kd in the reduced form. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine factor I can be purified from serum by a simple 4-step procedure. It is structurally and functionally comparable to factor I of other species, and its purification completes the isolation and characterization of all the soluble components of the bovine alternative complement pathway.

In vitro analysis of complement-dependent HIV-1 cell infection using a model system.

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.

Expression of the complement alternative pathway by human myoblasts in vitro: biosynthesis of C3, factor B, factor H and factor I.

In this study, we demonstrate expression in vitro of complement alternative pathway components C3, factor B, factor H and factor I by normal human myoblasts and human rhabdomyosarcoma cell lines CRL1558 and HTB153. Proteins in culture supernatants were detected by Western (protein) blot analysis and biosynthetic labeling followed by immunoprecipitation experiments, and quantified by ELISA. Newly secreted proteins were structurally and functionally similar to their serum counterparts. An additional polypeptide of 43 kDa with factor H immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Protein expression was correlated with mRNA expression by reverse transcriptase-polymerase chain reaction analysis. The major proteins of complement alternative pathway C3, factor B and factor H were produced constitutively by skeletal muscle cells at a rate of 50 to 150 ng/10(6) cells/ml and factor I was expressed 20 ng/10(6) cells/ml. These syntheses in vitro were regulated by inflammatory cytokines. Interferon-gamma significantly upregulated C3, factor B and factor H expression, but had no effect on factor I production. Interleukin-1 beta strongly enhanced C3 and factor B production and had a weak enhancing or no effect on factor I and factor H secretion. Human myoblast cell lines constitute an interesting model to analyze complement biosynthesis by human skeletal muscle cells. Local complement expression by skeletal muscle in vivo may be implicated in some muscular inflammatory or pathological processes.

The fractionation of human plasma proteins. III. Purification of complement factors D and I using affinity chromatography.

A purification protocol for complement factors D and I was designed as part of a larger scheme for the purification of more than 20 human plasma proteins. The key affinity step in the purification of both serine proteases, factors D and I, was affinity chromatography using benzamidine Sepharose. The recoveries were 19 and 12% for factors D and I, respectively.

Molecular modelling of the domain structure of factor I of human complement by X-ray and neutron solution scattering.

Factor I is a typical multidomain protein of the complement system. It regulates complement activation by proteolytic degradation of C3b or C4b in the presence of factor H, complement receptor type 1, membrane cofactor protein or C4b-binding protein as cofactor. It is constructed from five presumed independently folded domains, namely a factor I module, a CD5-like domain, two low-density-lipoprotein receptor type A domains and a serine-proteinase domain. X-ray and neutron solution scattering was used to study the arrangement of these domains in factor I. Factor I was determined to be monomeric in solution, with an A280(1%,1cm) of 12.3-14.1. Its radius of gyration (RG) was 3.96 nm by X-rays in a high positive solute-solvent contrast, and 3.84 nm by neutrons at infinite solute-solvent contrast. The cross-sectional radius of gyration (RXS) was likewise found to be 1.64 nm by X-rays and 1.55 nm by neutrons. The RG data were not noticeably dependent on the solute-solvent contrast, whereas the RXS data showed a small dependence. The maximum dimension of factor I was determined to be 12.8 nm from the RG and RXS data, and 14-15 nm from the X-ray and neutron distance distribution functions. This length is too short to account for a linear arrangement of the domains in factor I. Small sphere models were developed for factor I in which the largest domain was modelled from the crystal structure for beta-trypsin. The attachment of either an elliptical cylinder or a two-armed V-shaped structure to this domain to represent the remaining four small domains gave good scattering curve-fits for factor I, and were compatible with experimental sedimentation coefficients. The non-extended domain models for factor I imply that the steric accessibility of each domain will be reduced, and this may be important for its functional activity.