Bovine C1q Research Articles

Deposition of complement components on Streptococcus agalactiae in bovine milk in the absence of inflammation.

Bovine milk is generally considered to be almost devoid of complement, on the basis of undetectable hemolytic activity, unless inflammation recruits plasma components. This study examines the deposition of complement components on a mastitis-causing isolate of Streptococcus agalactiae by enzyme-linked immunosorbent assay (ELISA). Neat milk from mid-lactating, uninflamed mammary glands (normal milk) effected marked C3 deposition on bacteria. Kinetic studies showed a protracted lag period (30 to 45 min) preceding C3 deposition, which required about 2 h to reach a maximum. Experiments with diluted serum suggested that this slow C3 deposition resulted mainly from the low concentration of certain components of complement in milk. Bacteria incubated in neat milk readily bound bovine conglutinin, indicating the presence of iC3b. Elution of covalently bound C3 fragments with hydroxylamine confirmed the deposition of C3b and iC3b on bacteria. Deposition of C4 on bacteria was not detected in neat milk, suggesting that C3 deposition did not result from the activation of the classical pathway. This was not the result of a lack of antibodies. However, C4 deposition could be obtained by adding purified bovine C1q to normal milk, and C3 deposition was accelerated, suggesting the participation of the classical pathway. The deposition of C1q on antibody-sensitized bacteria was impeded by milk compared with that of C1q diluted in phosphate-buffered saline. Concentrations of C1q in normal milk were very low, ranging from 150 to 250 ng/ml. Overall, these findings indicate that C1q was a limiting factor of the classical pathway in normal milk. The capacity of milk to deposit C3 fragments on mastitis-causing S. agalactiae prompts further studies to investigate its role in opsonophagocytosis.

Role of liver endothelial and Kupffer cells in clearance of human C1q in rats.

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) C1q in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu C1q were measured. In untreated rats, the clearance of hu C1q occurred in a monophasic manner with a half-life of 66 +/- 26.7 min. The clearance of hu C1q in KC-depleted rats was delayed significantly (p < 0.001) and occurred with a half-life of 217 +/- 78.8 min. Fifteen min after injection, 11 +/- 3.5% of hu C1q was found in the liver of untreated rats and 8 +/- 1.4% was found in the liver of KC-depleted rats. The percentage non-trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of C1q, reached a level of 11.6 +/- 5.6% at 240 min in untreated rats compared with 4.6 +/- 5.8% in KC-depleted rats. Double immunofluorescence staining 5 min after administration of C1q in untreated rats, revealed that C1q was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu C1q, there was an uptake of C1q in the hepatocytes. In KC-depleted rats, 5 min after administration of hu C1q, C1q was bound to the EC. Fifteen minutes after injection, C1q was also found in the hepatocytes. Electron microscopical studies revealed that C1q binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu C1q in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade C1q, and that EC may play a role in the handling of C1q and C1q bound to immune complexes.

C1q, a collagen-like complement subcomponent, in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.

Antigenic interspecies cross-reaction of subcomponent C1q of the first component of complement: evidence for its cross-reaction in both collagen-like and non-collagen-like regions.

Human, bovine, and mouse C1q, a subcomponent of the first complement component, were purified, and both globular (GF) and collagen-like fragments (CLF) were isolated from human and bovine C1q. Antisera were produced in rabbits with these C1q or fragments, and F(ab')2 of immunoglobulin G (IgG) was purified from the antisera in order to avoid the possible non-specific binding of C1q of these animals to the Fc portion of rabbit IgG. Immunodiffusion analyses and radioimmune inhibition tests with these F(ab')2 showed that the definitive antigenic cross-reactivity was among C1q molecules of these animals, and that the regions participating in interspecies cross-reactions were located in both GF and CLF of C1q. These results suggest that both the C-terminal non-collagenous globular and the N-terminal collagen-like domains of C1q molecules may have remained highly conserved during evolution.

An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. coli beta-galactosidase in the probe and C1q as the recognition unit

An enzymatically active probe (β-galactosidase-anti-β-galactosidase complex) is used to measure circulating immune complexes (CIC), in a competition assay where probe and CIC are confronted with a ‘recognition unit’. The latter is bovine conglutinin in the original description of this method. Here we describe a version utilizing human or bovine C1q. The two techniques are compared for their sensitivity and specificity, on both in vitro formed tetanus toxoid-anti-toxoid complexes and on sera from patients with selected diseases. The results confirm that the two recognition units are sensitive to families of CIC that only partially overlap. The parallel use of conglutinin and C1q yields both quantitative and qualitative information on the nature of CIC in individual sera.

Structures of the asparagine-linked sugar chains of subcomponent C1q of the first component of bovine complement.

Bovine C1q, a subcomponent of the first component of complement, contains six asparagine-linked sugar chains in 1 molecule. The sugar chains are exclusively distributed in the noncollagenous regions. The sugar chains were liberated as radioactive oligosaccharides from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction, and their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Bovine C1q was shown to contain equal amounts of neutral and acidic oligosaccharides with the following structures: (formula, see text) where NeuG1 is N-glycolylneuraminic acid.

Use of Bovine C1q to Detect Plant Viruses in an Enzyme-linked Immunosorbent-type Assay

SUMMARY Polystyrene microtitre plates coated with bovine C1q were used to trap antigen: antibody aggregates of three plant viruses [plum pox virus (PPV), potato virus Y and cocksfoot mild mosaic virus] and human antibody aggregates in an ELISA-type system. The aggregates were detected with alkaline phosphatase-labelled anti-rabbit or anti-human IgG. The assay was as sensitive as the double antibody sandwich method of ELISA for the detection of PPV particles; the limit of detection was 4 to 15 ng virus/ml in purified preparations or a dilution of &gt; 1/1000 in infective Nicotiana clevelandii sap extracts. When used at low dilutions, plant sap of three of the four species tested had an inhibitory effect on the reaction.

Purification and characterization of subcomponent C1q of the first component of bovine complement

Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.