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J E Volanakis

Publications and source records attributed to J E Volanakis.

At least 55 records · Page 3Linked to original sources
PubMed1995

Catalytic role of a surface loop of the complement serine protease factor D.

We have investigated the structural determinants of the unique functional properties of complement factor D by constructing and testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binding pocket of factor D with that of trypsin resulted in a mutant (M1) with greatly reduced proteolytic activity and slightly reduced reactivity toward small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic and esterolytic activities of factor D, produced a mutant (M2) with reactivities similar to M1. Replacement of the surface loop formed by residues 184-188 of M1 and M2 with the corresponding loop of trypsin produced mutants exhibiting one and two orders of magnitude higher esterolytic activity, respectively, than native factor D. However, the proteolytic activity of both mutants was similar to that of M1 and M2. We conclude that loop184-188 is an important determinant of the geometry of the primary specificity pocket of factor D. The low proteolytic activity of these mutants supports the proposal that the proteolytically active conformation of factor D is induced by its natural substrate, C3bB.

Amino Acid Sequence↗
PubMed1995

Transcriptional regulation of complement genes.

Complement is a major effector system of host defense against invading pathogens. The recently completed cloning and structural characterization of almost all complement genes has allowed investigation of their regulation at the molecular level. Transcription of most complement genes is accelerated during the acute-phase response that follows tissue injury. Mechanisms regulating transcription of other acute-phase proteins have been elucidated during recent years. The main mediators of acute phase proteins are IL-1- and IL-6-type cytokines. These cytokines and IFN gamma induce transcription of complement genes in the liver and in several extrahepatic sites. Consensus elements binding transcription factors activated by these cytokines have been identified in the promoters of several complement genes. However, only a few complement promoters have been characterized functionally. Structural analysis has indicated that TATA-less promoters are common among complement genes. However, no shared initiator elements (Inr) have been identified so far.

Acute-Phase Reaction↗
PubMed1994

Mutational analysis of the substrate binding site of human complement factor D.

Complement factor D is a serine protease with a single natural substrate, C3b-complexed factor B, and very low catalytic activity against synthetic esters. The recently solved X-ray crystal structure of factor D has demonstrated certain key differences from other serine protease in the conformation of residues of the catalytic triad and the substrate-binding regions. To investigate possible contributions of unique amino acid substitutions to these distinct structural and functional features of factor D, we constructed a series of mutants by substituting trypsin substrate-binding residues for the corresponding factor D residues. Wild-type and seven mutant factor D cDNAs were expressed stably in Chinese hamster ovary cells, and the recombinant proteins were purified from culture supernatants and assayed by hemolytic, proteolytic, and esterolytic assays. The combined results indicate that residues Thr-198, Ser-199, Arg-202, and perhaps also Val-203 provide determinants for substrate binding and catalysis. The data also provide additional support for the hypothesis that the proteolytically active conformation of the active center of factor D is induced by its substrate, C3bB.

Amino Acid Sequence↗
PubMed1994

Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis.

We have used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the C1q-binding site of C-reactive protein (CRP). Eleven mutant rCRP cDNAs, D112N; D112A; D112K; D112E; K114T; K114A; K114E; K114R; R116L; D112N, K114T; and D112N, R116L were constructed and expressed in COS cells. Wild-type (wt) and all mutant rCRPs bound to phosphocholine-substituted BSA and also to pneumococcal C-polysaccharide with apparent avidities similar to native CRP, except for the R116L mutant which bound slightly less avidly. Substitution of Asn, Ala, or Lys for Asp-112 resulted in decreased avidity of ligand-bound CRP for C1q and also in decreased C-activating efficiency as estimated from a C3-fragment deposition assay. However, complexes of the D112E mutant reproducibly bound better to C1q and activated the classical pathway more efficiently than wt rCRP. Substitution of Thr, Ala, or Glu for Lys-114 increased the avidity for C1q by 2- to 3-fold and the efficiency of classical pathway activation by 20- to 30-fold compared with wt CRP. In contrast, the K114R mutant was only slightly different from wt CRP. Substitution of Leu for Arg-116 did not significantly affect C1q-binding but resulted in increased C-activating efficiency. The data indicate that the negative charge of residue Asp-112 plays a major role in the formation of the C1q-binding site of CRP and that the positively-charged residue Lys-114 and to a lesser extent also Arg-116 play important but indirect roles in C1q-binding and activation of C by CRP complexes.

Amino Acid Sequence↗
PubMed1994

Recombinant and native zymogen forms of human complement factor D.

We have expressed a full-length cDNA clone encoding human factor D by using a baculovirus expression system. The purified recombinant protein reacted with Ab against native factor D, but was hemolytically inactive and slightly larger than factor D. These results suggested that the recombinant protein was the elusive zymogen of factor D. Amino acid sequencing demonstrated that the recombinant factor D consisted of two proenzyme forms with respective activation peptides, AAPPRGR and APPRGR. Catalytic amounts of trypsin converted recombinant profactor D to its enzymatically active form, exhibiting SDS-PAGE mobility and specific hemolytic activity similar to those of native factor D. About 90% of trypsin-activated recombinant profactor D had the same NH2-terminus as factor D. Human thrombin, kallikrein, and plasmin could also activate recombinant profactor D, but relatively high concentrations of these enzymes were required and the specific hemolytic activity of the "activated" profactor D was about one-third that of native factor D. Trypsin-activatable profactor D was also purified from the urine of a patient with Fanconi's syndrome. This native profactor D represented less than 1.0% of the total antigenic factor D in the patient's urine and had a Gly-Arg dipeptide as the activation peptide. Apparently, urine profactor D was produced by cleavage of pre-profactor D at Arg-(-3) by a serine protease with trypsin-like specificity, which probably is different from the putative leader peptidase that produces the recombinant profactor D. Urine profactor D was inhibited by diisopropyl fluorophosphate although the recombinant proenzyme was resistant to this inhibitor.

Amino Acid Sequence↗
PubMed1994

Alternatively spliced transcripts of the human complement C2 gene.

To investigate the possible presence of alternatively spliced C2 gene transcripts, we amplified mRNA from HepG2 cells by reverse transcription-PCR using primers derived from the 5' and 3' untranslated regions of the C2 mRNA. Cloning of the resulting products revealed the presence of four novel C2 mRNA size variants. Nucleotide sequencing indicated that the variant mRNAs were probably derived through differential splicing of C2 gene transcripts. Specifically, nucleotide sequence deletions in the four variant mRNAs could be attributed to splicing out of: 1) exons 2 and 3; 2) exon 3; 3) exon 17; and 4) exons 6 and 7 and the 5' region of exon 18. The results were confirmed by RNase protection assays using HepG2 mRNA. Inspection of the nucleotide and the deduced amino acid sequences indicated that in the first two variants the alternative splicing did not affect the C2 open reading frame. In the other two variants, frameshifts in exon 18 resulted in termination codons up- or downstream of the authentic termination codon. All four variants C2 mRNAs were capable of encoding truncated C2 proteins, and were detected by reverse transcription-PCR not only in HepG2 cells but also in human liver, U937, and U105-MG cells. The latter analyses indicated the presence of an additional C2 mRNA variant lacking the region encoded by exon 6.

Alternative Splicing↗
PubMed1994

Preliminary crystallographic studies on human complement pro-factor D.

The recombinant zymogen of the human complement protein factor D has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 as precipitant. Two crystal forms obtained at pH 5.4 belong to space group P2(1). The crystals grow to dimensions of 0.6 mm x 0.3 mm x 0.3 mm in three days, are stable in the X-ray beam, and diffract to 2.4 A.

Amino Acid Sequence↗
PubMed1994

Structure of human factor D. A complement system protein at 2.0 A resolution.

Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The present model was refined to an R-factor of 18.8% using 23,681 observed reflections between 7.5 and 2.0 A resolution, with a root-mean-square deviation from standard bond lengths of 0.016 A. The two non-crystallographically related molecules in the triclinic unit cell have distinctive active site conformations. The protein has the general structural fold of a serine protease, but there are several unique amino acid substitutions resulting in significant alterations in the critical loops responsible for catalysis and substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-dimensional structure has been determined.

Amino Acid Sequence↗
PubMed1994

Ancestry of SINE-R.C2 a human-specific retroposon.

We have reported previously that a retroposon, containing a variable number of tandemly repeated nucleotide sequences, is present in the third intron of the human C2 gene. This element, termed SINE-R.C2, is a member of a large retroposon family derived from the endogenous retrovirus HERV-K10 and estimated to include a few thousand copies per haploid human genome. In the present study we analyzed genomic DNA from 175 humans from several ethnic groups including Americans of European and African descent, Chinese, Africans, Australians, Pacific Islanders, Japanese, and Koreans. They all contained SINE-R.C2, as indicated by Southern blotting. However, SINE-R.C2 was absent from the genome of nonhuman primates, although SINE-R-type elements were present in chimpanzees and gorillas and the HERV-K10 genome was apparently present in all primates except for New World monkeys. These results indicate that HERV-K10 was inserted into the genome after the divergence of New World monkeys; the prototype SINE-R element, after divergence of orangutans; and SINE-R.C2, after the split between humans and chimpanzees.

Animals↗
PubMed1993

Modelling of the serine-proteinase fold by X-ray and neutron scattering and sedimentation analyses: occurrence of the fold in factor D of the complement system.

Solution scattering is a powerful means of determining the overall arrangement of domains in the multidomain proteins of complement. the serine-proteinase domain is central to all proteolytic events during complement activation. As models of this domain, bovine beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A were studied by neutron and X-ray synchrotron solution scattering. At pH 7, all the X-ray and neutron M(r) values corresponded to monomeric proteins. The X-ray radii of gyration, RG, of beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A (measured in positive solute-solvent contrasts) were 1.59 nm, 1.78 nm, 1.71 nm and 1.76 nm (+/- 0.05-0.11 nm) in that order. Neutron contrast variation showed that the RG at infinite contrast, RC, for these four proteins were 1.57 nm, 1.70 nm, 1.67 nm and 1.78 nm (+/- 0.03 nm) in that same order. The radial inhomogeneity of neutron-scattering density, alpha, was positive at (5-13) x 10(-5), and corresponds to the preponderance of hydrophilic residues near the protein surface. On trypsinogen activation, a small reduction in the RG value of 0.13 +/- 0.07 nm was just detectable, while the RG of chymotrypsinogen A was unchanged after activation. The RC and alpha values of the four proteins can be calculated by using crystallographic co-ordinates. The reduced RG of beta-trypsin relative to trypsinogen was explained in terms of the removal of the extended N-terminal hexapeptide of trypsinogen. The full X-ray and neutron-scattering curves in positive and negative contrasts agreed well with scattering curves calculated from crystallographic coordinates to a nominal structural resolution of 4.5 nm, provided that the internal structure was considered in neutron modelling, and that the hydration was considered in X-ray modelling. Sedimentation-coefficient data also provide information on the disposition of domains in multidomain proteins. It was found that the hydrated X-ray sphere model could be directly utilized to calculate sedimentation coefficients. X-ray scattering on factor D showed from its RG of 1.78 nm that this is monomeric and very similar in structure to beta-trypsin. The X-ray-scattering curve of factor D was readily modelled using the beta-trypsin crystal structure after allowance for sequence changes. The success of these modellings provides a basis for the constrained modelling of solution scattering data for the multidomain proteins of complement.

Chymotrypsin↗
PubMed1993

cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex.

The RD gene, initially defined in the mouse, has been mapped between the Bf and C4A genes in the human major histocompatibility complex class III region. Using the mouse cDNA as a probe, we isolated and sequenced human RD cDNA clones. The composite nucleotide sequence consisted of 1301 nucleotides, excluding a poly(A) tail at the 3' end. It contained a single open reading frame encoding a polypeptide of 380 amino acid residues with a calculated molecular mass of 42274 Da. The most striking structural feature of the deduced amino acid sequence is a region consisting entirely of 24 tandem repeats of an Arg-Asp (or Glu) dipeptide. The human RD cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and used to produce antisera in rabbits. Western blot analysis and immunoprecipitation of lysates of biosynthetically labelled HeLa cells indicated that RD is a 44 kDa nuclear protein.

Amino Acid Sequence↗
PubMed1993

Structure of the human C2 gene.

A human genomic cosmid clone, S22A, was used to characterize the structure of the human C2 gene. Eighteen exons spanning 18 kb of DNA were mapped by nucleotide sequencing of exon-containing subclones and by Southern blotting. Introns vary in length between 83 bp and 4.4 kb and all intron/exon boundaries follow the AG/GT consensus rule for splicing. Exon 1 encodes 270 bp of the 5'-untranslated region of the C2 cDNA C2HL5-3 and 15 amino acids of the leader peptide. The three short consensus repeats of C2b are encoded by the single exons 2, 3, and 4, respectively. The von Willebrand factor type A-like domain of C2a is encoded by exons 6-10 and the serine protease domain by exons 11-18. Exons 5 and 15 appear to be unique to the C2 and factor B genes.

Amino Acid Sequence↗
PubMed1993

Human complement factor B: cDNA cloning, nucleotide sequencing, phenotypic conversion by site-directed mutagenesis and expression.

A full-length cDNA clone, BHL4-1, encoding factor B was isolated from a human liver cDNA library and sequenced in its entirety. It consists of 2388 bp which include a 5'-untranslated region of 40 bp, a single open reading frame, 2292 bp in length, and a 3'-untranslated region of 56 bp followed by a poly-A tail. The deduced amino acid sequence comprises 25 residues of a putative leader peptide and 739 residues of the mature polypeptide chain of the F allele of factor B. We constructed an S allele-like Q7R mutant of BHL4-1 by site-directed mutagenesis. Both the wild-type and mutant factor B cDNA were expressed transiently in a eukaryotic system. The specific hemolytic activities of the two recombinant factor B alleles and of native B were not significantly different from each other.

Alleles↗
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↗