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J E Volanakis

Publications and source records attributed to J E Volanakis.

At least 109 records · Page 6Linked to original sources
PubMed1985

Renal filtration and catabolism of complement protein D.

Complement protein D, a serine protease participating in the formation of the C3 convertase of the alternative complement pathway, has the lowest molecular weight (23,750) and serum concentration of all complement proteins. In normal serum, D is the rate-limiting protease of the alternative pathway of complement activation. We report that the serum concentrations of D in 20 patients with chronic renal failure (mean +/- S.D., 0.42 +/- 0.28 mg per deciliter) and in 16 patients on long-term dialysis (1.53 +/- 0.39 mg per deciliter) were significantly higher (P less than 0.001) than in 22 healthy adults (0.18 +/- 0.04 mg per deciliter). In chronic renal failure the serum concentration of D correlated with that of creatinine (r = 0.75, P less than 0.001). The serum concentrations of D found in patients with renal failure reached and in some cases exceeded those at which the protease is no longer rate-limiting. Thus, enhanced activity of the alternative pathway of complement should be expected in patients with advanced renal failure. Urinary D was undetectable (less than 0.2 micrograms per deciliter) in 17 normal adults and either undetectable or below the concentration expected from the degree of proteinuria in 10 patients with nephrotic syndrome. However, in a patient with Fanconi's syndrome the urinary concentration of D (1.3 mg per deciliter) was an order of magnitude higher than the serum concentration, representing 0.5 per cent of the total protein. The urinary D in this patient had normal hemolytic activity, antigenicity, and size. These results indicate that D is filtered through the glomerular membrane and is probably catabolized in the proximal renal tubules.

Adult↗
PubMed1985

Biosynthesis of complement protein D by HepG2 cells: a comparison of D produced by HepG2 cells, U937 cells and blood monocytes.

The biosynthesis of complement protein D of the alternative pathway by HepG2 cells, a human hepatocyte cell line, was studied and compared to the biosynthesis of D by U937 cells and blood monocytes. Increasing amounts of antigenic D were detected in HepG2 cell culture supernatants by radioimmunoassay. The kinetics of D synthesis and secretion by HepG2 cells was followed in a pulse-chase study using [35S]cysteine. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography, only a single D band was seen intra- and extracellularly and both forms had the same apparent molecular weight as D synthesized by U937 cells or purified from serum. Treatment of HepG2 and U937 cells with canavanine, an arginine amino acid analog, to inhibit intracellular processing resulted in slight depression of the apparent molecular weight of D synthesized by these cells. D synthesized by blood monocytes had an apparent molecular weight similar to that synthesized by HepG2 and U937 cells, suggesting that these cell lines do not synthesize and process D differently than normal monocytes. The data demonstrate that the hepatocyte is a site of D synthesis and suggest that D is not synthesized as a precursor molecule.

Canavanine↗
PubMed1985

Carbohydrate composition of the second, third and fifth components and factors B and D of human complement.

The carbohydrate composition of the second, third and fifth components of human complement (C2, C3 and C5) and of factors B and D was determined employing gas-chromatographic and mass-spectrometric methods. C2 was found to contain 15.9% carbohydrate composed of fucose, galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (approximate molar ratio 1:4:9:9:4). N-acetylglucosamine and mannose (approximate molar ratio 1:4), amounting to 1.7% of the mass of the molecule, were the only monosaccharides detected in C3. C5 contained 3.8% carbohydrate composed of galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (approximate molar ratio 2:4:4-5:2). The carbohydrate moiety of B consisted of fucose, galactose, mannose, N-acetylglucosamine and N-acetylneuraminate (molar ratio 1:2:3:4:2). The total carbohydrate content of B was estimated at 8.6%. In addition to these monosaccharides, glucose (0.4-0.9%) was also detected in all preparations analysed. Glucose was the only sugar detected in D.

Carbohydrates↗
PubMed1985

C-reactive protein binds leishmanial excreted factors.

Excreted factors from Leishmania tropica and Leishmania donovani are precipitated by human and rabbit C-reactive protein. The reaction is calcium dependent and appears to be similar to that reported to occur between C-reactive protein and various galactans. The absence of phosphate and N-acetyl galactosamine suggests that the reaction is not the result of any similarity of the excreted factors to pneumococcal C-polysaccharide.

Antigens, Protozoan↗
PubMed1985

Opsonic properties of C-reactive protein. Stimulation by phorbol myristate acetate enables human neutrophils to phagocytize C-reactive protein-coated cells.

We examined phagocytosis of sheep erythrocytes passively sensitized with pneumococcal C-polysaccharide (E-PnC) and of E-PnC coated with C-reactive protein (E-PnC-CRP) by human polymorphonuclear leukocytes (PMN). PMN isolated from blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after stimulation with 12-O-tetradecanoylphorbol-13-acetate (PMA; 2 ng/ml), PMN ingested E-PnC-CRP efficiently with a mean phagocytic index (PI) of 99.5 +/- 4.8 (mean +/- SD, n = 11), and E-PnC to a lesser extent with a mean PI of 33.2 +/- 11.7 (mean +/- SD, n = 11). PMN that had adhered to PnC-coated glass and that were stimulated with PMA attached but did not ingest E-PnC-CRP. In contrast, PMN plated on E-PnC-CRP-coated glass and stimulated with PMA did not attach or ingest E-PnC-CRP. These data indicate that PMN can be induced to phagocytize PnC-CRP and that both PnC and CRP are required for ingestion. They also suggest that specific receptors for these ligands are expressed by stimulated PMN. Neither attachment nor phagocytosis of E coated with rabbit anti-E IgG (E-IgG) was affected by plating PMN on PnC or PnC-CRP. On the other hand, both phagocytosis and ingestion of E-PnC-CRP as well as E-IgG was blocked by plating PMA-stimulated PMN on immune complexes containing rabbit IgG. Inhibition experiments with the use of 3G8, a monoclonal antibody to the Fc gamma receptor of PMN, and human monomeric IgG1 demonstrated that attachment of E-PnC-CRP is mediated by receptors other than the Fc gamma receptors. These combined results indicated a nonreciprocal association between the putative CRP receptors and the Fc gamma receptors of stimulated PMN, resulting in the clearance of both types of receptors from the apical surface of PMN by antigen-immobilized rabbit IgG.

Animals↗
PubMed1985

In vitro biosynthesis of complement protein D by U937 cells.

Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.

Amino Acids↗
PubMed1984

Amino acid sequence of human D of the alternative complement pathway.

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.

Amino Acid Sequence↗
PubMed1984

Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay.

A sensitive solid-phase radioimmunoassay is described which quantitates human D to 1-2 ng/ml. The assay was used to measure the concentration of D in normal and acute-phase sera and sera from individuals with systemic lupus erythematosus. All 3 groups of sera had comparable levels of D with mean values of 1.8, 2.3 and 2.5 micrograms/ml, respectively. Also tested were sera decomplemented in vitro by activators of the classical and alternative pathways. The results indicated that D is not depleted by alternative or classical pathway activation. However, heat inactivation (56 degrees C, 30 min) of serum resulted in almost complete loss of antigenic D.

C-Reactive Protein↗
PubMed1984

The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.

Animals↗
PubMed1983

A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer.

Use of o-phthaldialdehyde to chemically reduce the newly generated amino termini responsible for the progressively increasing background during an extended amino acid sequence analysis in a liquid phase sequencer has been described. The results have been compared with Fluram blocking using apomyoglobin and rabbit C-reactive protein as standard and unknown samples, respectively.

Aldehydes↗
PubMed1983

Binding of human C4 to C-reactive protein-pneumococcal C-polysaccharide complexes during activation of the classical complement pathway.

Sequential interaction of CRP-PnC aggregates, made at slight CRP excess, with purified human C1, C4 and C2oxy resulted in formation of an effective C3-convertase, indicating the binding of C1, C4 and C2 on the aggregates. Immunoprecipitation experiments demonstrated that, following cleavage of 125I-C4 by CRP-PnC-C1 complexes, approximately 3% of the 125I-C4 was bound to CRP while a lower percentage was bound to PnC, CRP-C4 complexes could also be demonstrated by substituting 125I-CRP for 125I-C4. The nature of the CRP-C4 bond was examined by electrophoretic analysis. Complexes of 125I-C4-CRP prepared as earlier were incubated at 100 degrees C for 2 min in buffer containing 2% SDS and 5% beta-mercaptoethanol and subjected to electrophoresis in SDS-containing polyacrylamide gradient slab gels. Autoradiography of the dried gels revealed the presence of high mol. wt bands containing the alpha'-chain of C4b. CRP could also be demonstrated in these high mol. wt bands which apparently represented covalent complexes between the alpha'-chain of C4b and CRP monomers. Since CRP contains no detectable carbohydrate, it seems likely that an amide bond is formed between the two proteins.

Amides↗
PubMed1983

Cytomegalovirus-infected cell polypeptides immune-precipitated by sera from children with congenital and perinatal infections.

Congenital or perinatally acquired human cytomegalovirus (CMV) infections in children may be symptomatic or asymptomatic. In this study, we characterized the electrophoretic properties of CMV-infected cell polypeptides immune-precipitated by sera from children with different types of CMV infections from birth to 4 years of age. Sodium dodecyl sulfate-polyacrylamide gel analysis of immune precipitates formed with radiolabeled extracts of cells infected with CMV strain AD169 showed the following. (i) Electrophoretic profiles of CMV polypeptides immune-precipitated by sera from children with perinatal and congenital infections were similar. At least 11 polypeptides with apparent molecular weights of 150,000, 140,000, 110,000, 100,000, 74,000, 66,000, 50,000, 49,000, 34,000, 25,000, and 20,000 were precipitated. Antibody titer in anticomplement immunofluorescence tests and virus titer in urine correlated with the intensity of polypeptide profiles in autoradiograms. (ii) The initial immune response of children with symptomatic congenital infections was delayed as compared to that of children with asymptomatic congenital and perinatal CMV infections. Sera obtained serially from symptomatic children for years after birth continued to precipitate CMV polypeptides, whereas sera from children with subclinical congenital infections precipitated lesser amounts over time. (iii) Immune precipitates obtained with sera from CMV-infected patients and with monoclonal antibodies to CMV contained polypeptides with comparable electrophoretic and immunological properties.

Antibodies, Monoclonal↗
PubMed1983

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy.

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.

Animals↗
PubMed1982

Demonstration of calcium-induced conformational change(s) in C-reactive protein by using monoclonal antibodies.

Three out of four anti-C-reactive protein monoclonal antibodies [HB3-2, (micro, k)], EA4-1 (gamma 2a, k) and FB2-1 (gamma 1, k) bind to C-reactive protein in the presence of 2.5 mM Ca2+ but not in the presence of 1.0 mM EDTA, indicating that the conformation of the antigenic determinant(s) recognized by these three antibodies is dependent upon Ca2+. This Ca2+-dependent binding can be inhibited by 1.0 mM phosphocholine, indicating that this antigenic determinant is at or near the phosphocholine-binding site of C-reactive protein. The binding of the fourth monoclonal antibody [HD2-4 (gamma 2-k)] is independent of the presence of Ca2+ and is not inhibited by phosphocholine. HB3-2 (micro, k) recognizes an antigenic determinant on the structurally related proteins, rabbit CRP and serum amyloid P.

Antibodies, Monoclonal↗