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J E Volanakis

Publications and source records attributed to J E Volanakis.

At least 19 recordsLinked to original sources
PubMed1992

Probing the phosphocholine-binding site of human C-reactive protein by site-directed mutagenesis.

Human C-reactive protein (CRP) can activate the classical pathway of complement and function as an opsonin only when it is complexed to an appropriate ligand. Most known CRP ligands bind to the phosphocholine (PCh)-binding site of the protein. In the present study, we used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the PCh-binding site of CRP. Eight mutant recombinant (r) CRP, Y40F; E42Q; Y40F, E42Q; K57Q; R58G; K57Q, R58G; W67K; and K57Q, R58G, W67K were constructed and expressed in COS cells. Wild-type and all mutant rCRP except for the W67K mutants bound to solid-phase PCh-substituted bovine serum albumin (PCh-BSA) with similar apparent avidities. However, W67K rCRP had decreased avidity for PCh-BSA and the triple mutant, K57Q, R58G, W67K, failed to bind PCh-BSA. Inhibition experiments using PCh and dAMP as inhibitors indicated that both Lys-57 and Arg-58 contribute to PCh binding. They also indicated that Trp-67 provides interactions with the choline group. The Y40F and E42Q mutants were found to have increased avidity for fibronectin compared to wild-type rCRP. We conclude that the residues Lys-57, Arg-58, and Trp-67 contribute to the structure of the PCh-binding site of human CRP. Residues Tyr-40 and Glu-42 do not appear to participate in the formation of the PCh-binding site of CRP, however, they may be located in the vicinity of the fibronectin-binding site of CRP.

Amino Acid Sequence↗
PubMed1992

The proteoglycan decorin binds C1q and inhibits the activity of the C1 complex.

Decorin, a small collagen-binding dermatan sulfate proteoglycan, is widely distributed as a component of extracellular matrices. Using a solid phase binding assay, we showed that decorin bound C1q at physiologic pH and ionic strength. The interaction did not require divalent cations and was time and temperature dependent reaching equilibrium in 4 h at 37 degrees C. Binding was specific and saturable with an apparent dissociation constant of 7.6 x 10(-9) M. Decorin was shown to bind pepsin-derived fragments containing the collagenous domain of C1q and collagenase-derived fragments containing the globular domain of C1q. Because these fragments share a short sequence of amino acids, this finding suggests that decorin binds to a region of C1q located near the junction of the two domains. Competition studies using purified preparations of the decorin core protein and the glycosaminoglycan chains showed that only the former inhibited binding of decorin to C1q indicating that the interaction is mediated by the decorin core protein. Decorin was shown to inhibit the hemolytic activity of purified C1 as well as C1 in normal human serum. Approximately 50% inhibition was observed at a decorin concentration of 2 micrograms/ml. Inhibition was not observed if C1 was bound to Ag-complexed antibody. Furthermore, neither the core protein nor the glycosaminoglycan chain of decorin inhibited C1, indicating that the intact proteoglycan is necessary for functional activity.

Animals↗
PubMed1992

Production and interferon-gamma-mediated regulation of complement component C2 and factors B and D by the astroglioma cell line U105-MG.

In this paper, we demonstrate the synthesis of the complement component C2 and factors B and D by the human astroglioma cell line U105-MG. All three components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of complement proteins, and particularly of alternative pathway activation components (C3, factors B and D), may play an important role in host defence in the central nervous system.

Astrocytoma↗
PubMed1992

Substituted isocoumarins as inhibitors of complement serine proteases.

Inhibition of complement proteins D, B, C2, C1s, C1r, I, and the catalytic fragments Bb and C2a by substituted isocoumarins was investigated. 3,4-Dichloroisocoumarin, a general serine protease inhibitor, inhibited factor D, C1r, and C1s moderately with second-order inhibition constants (kobs/[I]) of 40 to 190 M-1 s-1, but it did not inhibit C2, factor B, C2a, or Bb. The best inhibitor for factors D and B was 4-chloro-7-guanidino-3-methoxyisocoumarin with kobs/[I] values of 250 and 290 M-1 s-1, respectively. Most isocoumarins did not inhibit C2 or C2a; only 4-chloro-3-isothiureidoalkoxyisocoumarins were slightly inhibitory. 3-Alkoxy-4-chloro-7-guanidinoisocoumarins inhibited C1r and C1s moderately. The best inhibitor for C1r and C1s was 4-chloro-3-(3-isothiureidopropoxy)isocoumarin with kobs/[I] values of 6,600 and 130,000 M-1 s-1, respectively. Fifty amino acid or peptide thioesters containing Arg or other amino acids at the P1 site were tested as substrates of factor I, however none was hydrolyzed. Isocoumarins substituted with chloro and basic groups such as guanidino and isothiureidoalkoxy inhibited factor I activity with its natural substrate C3b, but kobs/[I] values were low. 4-Chloro-3-ethoxy-7-guanidinoisocoumarin inhibited activation of the alternative pathway and, to a lesser extent, of the classical pathway in serum. Several other substituted isocoumarins also inhibited cobra venom factor-initiated activation of the alternative pathway in serum.

Amino Acid Sequence↗
PubMed1992

A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus.

We have previously described multiallelic restriction fragment length polymorphisms of the C2 gene, suggesting the presence of a variable number of tandem repeats (VNTR) locus. We report here the cloning and sequencing of the polymorphic fragments from the two most common alleles of the gene, a and b. The results confirm the presence of a VNTR locus consisting of a nucleotide sequence, 41 bp in average length, repeated tandemly 23 and 17 times in alleles a and b, respectively. The difference in the number of repeats between the two alleles is due to the deletion/insertion of two noncontiguous segments, 143 and 118 bp long, of allele a, and of a 40-bp segment of allele b. The VNTR region is associated with a SINE (short interspersed sequence)-type retroposon, SINE-R.C2, located within the third intron of the C2 gene. SINE-R.C2 is a member of a previously described large retroposon family of the human genome, apparently derived from the human endogenous retrovirus, (HERV) K10, which is homologous to the mouse mammary tumor virus.

Alleles↗
PubMed1992

Genetic and immunologic analysis of a family containing five patients with common-variable immune deficiency or selective IgA deficiency.

A family with 13 members included 2 subjects with selective IgA deficiency (IgA-D) and 3 subjects with common-variable immune deficiency (CVID), diseases which usually occur sporadically. Reciprocal combinations of B and T cells in vitro between one normal and two immune-deficient family members and normal subjects revealed that defective Ig synthesis was determined by the B cells, while the patient T cells functioned normally. Normal T helper and suppressor function was demonstrated even in one patient with CVID who developed a T-cell lymphoproliferative disorder associated with elevated IgM; this patient's B cells made only IgM in vitro. Immune deficiencies were inherited in this family in a pattern consistent with an autosomal dominant trait with incomplete penetrance. All the immune-deficient patients in this family possessed at least one copy of an MHC haplotype previously shown to be abnormally frequent in IgA-D and CVID: HLA-DQB1*0201, HLA-DR3, C4B-Sf, C4A-deleted, G11-15, Bf-0.4, C2-a, HSP70-7.5, TNF alpha-5, HLA-B8, and HLA-A1. The patient who developed the lymphoproliferative disorder was homozygous for this haplotype. Four immunologically normal members, one of whom was 80 years old, also possessed this MHC haplotype, indicating that its presence is not sufficient for disease expression. A small segment of another MHC haplotype associated with Ig deficiency in the population also occurred in this family, but it was not associated with immune deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Major histocompatibility complex class III genes and susceptibility to immunoglobulin A deficiency and common variable immunodeficiency.

We have proposed that significant subsets of individuals with IgA deficiency (IgA-D) and common variable immunodeficiency (CVID) may represent polar ends of a clinical spectrum reflecting a single underlying genetic defect. This proposal was supported by our finding that individuals with these immunodeficiencies have in common a high incidence of C4A gene deletions and C2 rare gene alleles. Here we present our analysis of the MHC haplotypes of 12 IgA-D and 19 CVID individuals from 21 families and of 79 of their immediate relatives. MHC haplotypes were defined by analyzing polymorphic markers for 11 genes or their products between the HLA-DQB1 and the HLA-A genes. Five of the families investigated contained more than one immunodeficient individual and all of these included both IgA-D and CVID members. Analysis of the data indicated that a small number of MHC haplotypes were shared by the majority of immunodeficient individuals. At least one of two of these haplotypes was present in 24 of the 31 (77%) immunodeficient individuals. No differences in the distribution of these haplotypes were observed between IgA-D and CVID individuals. Detailed analysis of these haplotypes suggests that a susceptibility gene or genes for both immunodeficiencies are located within the class III region of the MHC, possibly between the C4B and C2 genes.

Agammaglobulinemia↗
PubMed1991

Site-directed mutagenesis of the region around Cys-241 of complement component C2. Evidence for a C4b binding site.

We probed the functional significance of the region around Cys-241 in human C2 by testing the hemolytic activity of a series of mutant rC2. Mutant C2 cDNA were constructed by oligonucleotide-directed site-specific mutagenesis and expressed transiently in COS cells. Wild-type rC2 had threefold higher specific hemolytic activity than native serum C2. Substitution of Gly, Ala, or Ser for Cys-241 resulted in a slightly, but significantly, increased activity. In addition, I2 had no effect on the activity of these mutant C2. Substitution of Lys for Gln-243 increased the hemolytic activity by more than two-fold. Increased activity in all cases was due to slower decay rates of the C3 convertase. Finally, substitution of Leu or Ala for Asp-240 or Ser-244, respectively, resulted in more than 100-fold decrease of hemolytic activity. The results suggest that residues 240 to 244 of human C2 represent an important structural determinant of the C4b binding site of C2a. They also confirm that Cys-241 is the residue responsible for the increased activity of C2 reacted with I2.

Amino Acid Sequence↗
PubMed1991

Crystallization and preliminary X-ray investigation of factor D of human complement.

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.

Complement Factor D↗
PubMed1991

IgA deficiency.

IgA deficiency, the most common primary immunodeficiency, is a very heterogeneous clinical disorder which may be associated with a variety of infections, allergies, autoimmune disorders, gastrointestinal diseases, and genetic disorders. The central phenotypic feature of this immunodeficiency is a B cell differentiation arrest, the extent of which may determine the clinical variability. Integrity of the immunoglobulin genes and their expression by immature B cells in affected individuals suggests an immunoregulatory basis for the B cell arrest. Genetic studies imply that a susceptibility gene in or near the major histocompatibility locus may predispose homozygous individuals to a spectrum of antibody deficiencies which may range from isolated IgA deficiency to panhypogammaglobulinemia. Essential cofactors in the pathogenesis of IgA deficiency include environmental factors, such as certain drugs and viral infections.

B-Lymphocytes↗
PubMed1990

Rotation function studies of human C-reactive protein.

Rotation function studies of two tetragonal crystal forms of human C-reactive protein have confirmed the pentameric structure of the molecule. The two crystal forms have space groups P4122 (I) and P4222 (II) with closely similar unit cells and are often twinned together. Investigation of the crystallization conditions indicates that dissociation heterogeneity has been a major limiting factor in the reproducible growth of good single crystals. The orientation of the pentameric molecule is shown to be almost identical in both forms, about the axial direction omega = 57 degrees, phi = 45 degrees, i.e. 57 degrees away from c in the (110) plane.

C-Reactive Protein↗
PubMed1990

Allelic associations of multiple RFLPs of the gene encoding complement protein C2.

The gene for the second component of complement, C2, maps within the class III region of the major histocompatibility complex (MHC). Many human diseases have been reported to be associated with MHC alleles, haplotypes, or extended haplotypes, but in most cases additional polymorphic markers are needed for the eventual localization of the genes responsible for these diseases. In this study, nine C2 haplotypes for four restriction-site polymorphisms, detected by SstI, BamHI, and TaqI, were defined among 143 unrelated individuals. Two of these polymorphisms are multiallelic and map near the 5' end of the C2 gene. The extensive allelic variation of the C2 gene may prove of value in studies of diseases associated with the MHC.

Alleles↗
PubMed1990

[Quantitation of intact form of complement protein B and its fragment Ba in human serum by radioimmunoassay].

A sensitive solid-phase radioimmunoassay for intact complement protein B and its smaller fragment Ba is described which quantitates human B and Ba to 30 ng/ml. The assay was proved to be useful to evaluate the activation of the alternative pathway of the complement system by measuring intact B and Ba in normal human sera activated with zymosan. In sera from patients with systemic lupus erythematosus (SLE), the level of intact B was decreased and that of Ba was increased in the active phase, and the level of Ba was increased though intact B which was within normal limits in the inactive phase. The present result suggests that the activation of the alternative pathway is occurring both in the active and inactive phase in SLE, and the measurement of intact B and Ba concentration in pathological sera would be useful to evaluate the activation of the alternative complement pathway.

Adult↗
PubMed1979

Interaction of C-reactive protein with artificial phosphatidylcholine bilayers.

C-Reactive protein (CRP), the most characteristic of the 'acute phase proteins' (ref. 1) is thought to participate in the mediation and/or modulation of acute inflammatory processes, but its exact function is unknown. CRP has a Ca2+-dependent binding specificity for phosphorylcholine, the polar head group of two widely distributed lipids, lecithin (phosphatidylcholine, PC) and sphingomyelin (SM). A number of observations suggest that at least some of the biological activities of CRP depend on its interaction with phospholipids of cell membranes. In addition, interaction of CRP with PC- and SM-containing lipid dispersions and with PC-containing liposomes can activate the complement system. We report here that binding of CRP to model membranes of PC requires the incorporation into the bilayer of lysophosphatidylcholine (LPC). Thus, a disturbance of the molecular organisation of the bilayer appears to be necessary for binding of CRP. These findings provide a possible biochemical explanation for binding of CRP to damaged but not intact cell membranes and might be relevant to its biological function.

Binding Sites↗
PubMed1979

Immune complexes in rheumatoid arthritis sera and synovial fluids: a comparison of three methods.

Sera and synovial fluids from 88 patients with rheumatoid arthritis were examined for circulating immune complexes by three assays: monoclonal rheumatoid factor radioimmunoassay, C1q binding assay, and Raji cell radioassay. Paired samples were available for 82 patients. Immune complexes were detected with high frequency in the synovial fluid by each assay (75% by the monoclonal rheumatoid factor radioimmunoassay, 95% by the C1q binding assay, and 61% by the Raji cell readioassay). In rheumatoid arthritis sera, immune complexes were detected with high frequency by the C1q binding assay (85%) and the monoclonal rheumatoid factor radioimmunoassay (70%) but infrequently by the Raji cell radioassay (26%). The presence of immune complexes in serum was most frequently accompanied by the presence of complexes in fluid, regardless of the method of detection; moreover, the levels of immune complexes in synovial fluid were generally higher than in paired serum. Further, the levels of immune complexes as measured by the C1q binding assay correlated with certain parameters of clinical activity, while the monoclonal rheumatoid factor radioimmunoassay and Raji cell radioassay correlated with extraarticular features (excluding nodules) of rheumatoid arthritis.

Antigen-Antibody Complex↗
PubMed1979

Circulating immune complexes in mixed connective tissue disease.

Circulating immune complexes were measured by three assays (monoclonal rheumatoid factor radioimmunoassay, C1q-binding assay, Raji cell radioassay) in sera from patients with mixed connective tissue disease. Evidence for circulating immune complexes was found by at least one method in 94% of sera. The highest frequency of positive results was noted by the Raji cell radioassay (88%). However, a significant number of patients also had immune complexes measured by the C1q-binding assay (50%) and monoclonal rheumatoid factor radioimmunoassay (45%). Further, the levels of immune complexes are measured by the Raji cell radioassay, and to a lesser extent the C1q-binding assay, correlated with disease activity and represent a potentially useful clinical tool. The pattern of reactivity in mixed connective tissue disease sera differed from rheumatoid arthritis, scleroderma, and polymyositis. This suggests that a spectrum of immune complexes is found in these rheumatic diseases and supports the concept that mixed connective tissue disease is distinct from these diseases.

Antibodies, Antinuclear↗