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Biomedical subjects

J E Volanakis

Publications and source records attributed to J E Volanakis.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular defects leading to human complement component C6 deficiency in an African-American family.

Complement component C6 deficiency (C6D) was diagnosed in a 16-year-old African-American male with meningococcal meningitis. The patient's father and two brothers also had C6D, but gave no history of meningitis or other neisserial infection. By using exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism as a screening step and nucleotide sequencing of target exons, we determined that the proband was a compound heterozygote for two C6 gene mutations. The first, 1195delC located in exon 7, is a novel mutation, while the second, 1936delG in exon 12, has been described before to cause C6D in an unrelated African-American individual. Both mutations result in premature termination codons and C6 null alleles. Allele-specific PCR indicated that the proband's two brothers also inherited the 1195delC mutation from their heterozygous mother and the 1936delG mutation from their homozygous father.

Adolescent↗
PubMed1998

Evaluation of IgA deficiency in Sardinians indicates a susceptibility gene is encoded within the HLA class III region.

IgA deficiency (IgA-D) has been associated with the HLA region, in particular with the North European haplotype HLA-A1, -B8, -DR3, but the exact location of the susceptibility gene(s) is unknown. Some reports suggest that a susceptibility gene is encoded in the class II region, while others implicate the class III region. We exploited differences between the common Sardinian and North European HLA-DR3 haplotypes to help localize the IgA-D susceptibility gene(s). With the knowledge that approximately 13% of HLA-DR3 homozygous individuals of North European origin are IgA-D, we examined 43 HLA-DR3 homozygous Sardinians to find that all had normal serum IgA, IgG and IgM levels. A detailed analysis of their MHC haplotypes indicated a common Sardinian HLA-DR3 haplotype TAP1A, TAP2A, HLA-DQB1*0201, -DQA1*0501, -DRB1*0301, LH1-(Z + 2), D3A-(Z + 2), C4B-0, C4A-L, G11-15, Bf-0-4, C2-a, HSP70-7.5, 9N3-(Z + 10), 82I-(Z - 2), TNFalpha-9, 62-(Z - 20), HLA-B18, -Cw5, -A30 which diverges from the common North European HLA-DR3 haplotype telomeric to the HLA-DR region. In parallel studies of five Sardinians with IgA-D, two of the 10 HLA haplotypes (20%) contained HLA-DR3, a frequency similar to that observed in the background population. One of these was the HLA-DR3- B8 North European haplotype, which occurs rarely in Sardinia. Our data favour the hypothesis that a class III region allele, present on the common North European but not on the Sardinian HLA-DR3 haplotype, confers susceptibility to IgA-D.

Disease Susceptibility↗
PubMed1998

Susceptibility locus for IgA deficiency and common variable immunodeficiency in the HLA-DR3, -B8, -A1 haplotypes.

BACKGROUND: A common genetic basis for IgA deficiency (IgAD) and common variable immunodeficiency (CVID) is suggested by their occurrence in members of the same family and the similarity of the underlying B cell differentiation defects. An association between IgAD/CVID and HLA alleles DR3, B8, and A1 has also been documented. In a search for the gene(s) in the major histocompatibility complex (MHC) that predispose to IgAD/CVID, we analyzed the extended MHC haplotypes present in a large family with 8 affected members. MATERIALS AND METHODS: We examined the CVID proband, 72 immediate relatives, and 21 spouses, and determined their serum immunoglobulin concentrations. The MHC haplotype analysis of individual family members employed 21 allelic DNA and protein markers, including seven newly available microsatellite markers. RESULTS: Forty-one (56%) of the 73 relatives by common descent were heterozygous and nine (12%) were homozygous for a fragment or the entire extended MHC haplotype designated haplotype 1 that included HLA- DR3, -C4A-0, -B8, and -A1. The remarkable prevalence of haplotype 1 was due in part to marital introduction into the family of 11 different copies of the haplotype, eight sharing 20 identical genotype markers between HLA-DR3 and HLA-B8, and three that contained fragments of haplotype 1. CONCLUSION: Crossover events within the MHC indicated a susceptibility locus for IgAD/CVID between the class III markers D821/D823 and HLA-B8, a region populated by 21 genes that include tumor necrosis factor alpha and lymphotoxins alpha and beta. Inheritance of at least this fragment of haplotype 1 appears to be necessary for the development of IgAD/CVID in this family.

Adult↗
PubMed1997

Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras.

To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.

Antibodies, Monoclonal↗
PubMed1997

Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.

The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.

Amino Acid Sequence↗
PubMed1997

Structure of diisopropyl fluorophosphate-inhibited factor D.

Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limited number of general serine protease inhibitors are known to inhibit D, most of which covalently bind to the serine hydroxyl of the catalytic triad. The structure of the first enzyme:inhibitor covalent adduct of D with diisopropyl fluorophosphate (DIP:D) to a resolution of 2.4 A is described. The inhibited enzyme is similar in overall structure to the native enzyme and to trypsin, yet exhibits notable differences in the active site. One region of the active site is conserved between D and trypsin with respect to amino-acid sequence and to conformation. Another reflects the amino-acid substitutions and conformational flexibility between these enzymes. The active-site histidine residue is observed in the gauche+ conformation, not the normal gauche- orientation seen in the classic catalytic triad arrangement required for enzymatic activity in serine proteases. Comparisons of the active sites between native D, the DIP:D adduct, and DIP-inhibited trypsin have provided fundamental insights currently being employed in the design of novel small-molecule pharmaceutical agents capable of modulating the alternative complement pathway.

Journal Article↗
PubMed1997

Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67.

We have reported previously that residues Lys57, Arg58, and Trp67 of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site. In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared. Wild-type (wt) and all mutant CRPs were purified by affinity chromatography on PCh-, pneumococcal C-polysaccharide (PnC)-, or phosphoethanolamine-conjugated agarose columns. Purified mutant CRPs, K57Q/R58G/W67K and T76Y failed to bind to solid phase, PCh-substituted BSA. They did, however, bind to immobilized PnC, although with substantially decreased avidity compared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epitope. We conclude that Thr76 is a determinant of the PCh-binding site, probably interacting with the choline group. This conclusion is supported by recent crystallographic data indicating that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline. Trp67, Lys57, and Arg58 do not directly contact PCh, but appear to be required for the proper conformation of the binding site.

C-Reactive Protein↗
PubMed1997

C-reactive protein: structural biology, gene expression, and host defense function.

Over the past few years substantial insight was gained into the biology and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected with Streptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.

Acute-Phase Reaction↗
PubMed1997

Age-related changes in serum immunoglobulins in patients with familial IgA deficiency and common variable immunodeficiency (CVID).

The concentration of serum immunoglobulins in individuals with IgA deficiency (IgAD) and CVID can vary with age to have practical implications for evaluation, therapy, and genetic analysis. Most IgAD and CVID patients in our clinic population in the Southeastern United States have inherited part or all of two extended MHC haplotypes, referred to as haplotype 1 (HLA-DQB1 0201, HLA-DR3, C4B-Sf, C4A-0, G1-15, Bf-0.4, C2-a, HSP-7.5, TNF alpha-5, HLA-B8, HLA-A1) and haplotype 2 (HLA-DQB1 0201, HLA-DR-7, C4B-S, C4A-L, G11-4.5, Bf-0.6, C2-b, HSP-9, TNF alpha-9, HLA-B44, HLA-A29). In the present study, the clinic records of 68 CVID patients and 73 IgAD patients were reviewed to determine whether patients with familial or MHC-associated IgAD or CVID experience changes in serum immunoglobulin concentrations. An increase in serum immunoglobulin to the normal range was associated with clinical improvement in one patient with CVID and haplotype 2, two patients with IgAD and haplotype 2, and one IgAD patient whose haplotype was not determined. Two patients with haplotype 1 and one with haplotype 2 had a significant decline in serum immunoglobulin: one progressed from normal to IgAD associated with IgG subclass deficiencies, and two progressed from IgAD to CVID. Five of the seven patients with notable changing serum immunoglobulin levels have a family member with either IgAD or CVID. The findings suggest that familial, MHC-associated IgAD and CVID may be either progressive or reversible disorders, and emphasize the value of monitoring immunoglobulin levels in affected individuals and their family members.

Adolescent↗
PubMed1996

Genetic bases of human complement C7 deficiency.

Complement C7 deficiency (C7D) is associated frequently with recurrent bacterial infections, especially meningitis caused by Neisseria meningitidis. We report in this work the molecular bases of C7D in two unrelated Japanese males. We used exon-specific PCR/single-strand conformation polymorphism analysis as a screening step for mutations. Subsequent direct sequencing of the target exons identified homozygous mutations in exon 16 of case 1 and in exon 15 of case 2. The mutation of case 1 was a homozygous T to A transversion at nucleotide 2250, the third nucleotide of the codon TGT for Cys728, leading to a stop codon TGA (C728X). In case 2, a homozygous 2-bp deletion (2137delTG/2138delGT/2139delTG) caused a frameshift, generating a premature termination codon 4 to 6 nucleotides downstream. Family study in case 1 confirmed the genetic nature of the defect. Moreover, we detected a novel polymorphism in intron 11 that presumably is linked to the mutation responsible for C7D in case 1. Our results indicate that the pathogenesis of C7D is heterogeneous like most of the other deficiencies of complement components.

Adult↗
PubMed1996

Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.

We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.

Alternative Splicing↗
PubMed1996

Molecular bases for inherited human complement component C6 deficiency in two unrelated individuals.

Deficiency of the sixth component of complement (C6D) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular bases of C6D in two unrelated subjects, one African American (case 1) and the other Japanese (case 2). Screening all 17 exons of the C6 gene and their boundaries by exon-specific PCR/single strand conformation polymorphism demonstrated aberrant single stranded DNA fragments in exon 12 of case 1 and exon 2 of case 2. Nucleotide sequencing of the amplified DNA fragments revealed a homozygous single-base deletion (G1936) in exon 12 case 1 and a heterozygous single base deletion (C291/C292/C293/C294) in exon 2 of case 2. Both mutations resulted in frame shifts and premature termination of the C6 polypeptide. Sequence-specific oligonucleotide probe hybridization and direct sequencing of exon 12 amplified from genomic DNA further supported the homozygosity of the mutation in case 1. Case 2 is apparently compound heterozygote, but the putative mutation in the other allele of the C6 gene remains unknown. Both case 1 and case 2 were homozygous for the C6A allotype. These data indicate that at least three distinct mutational events can cause C6D, single nucleotide deletions in exons 2 and 12, and a mutation yet unidentified. Thus, similar to other complement protein deficiencies, the pathogenesis of C6D appears to be heterogeneous.

Adult↗
PubMed1996

Complement factor D, a novel serine protease.

Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.

Amino Acid Sequence↗
PubMed1996

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

Amino Acid Sequence↗
PubMed1996

Role of complement in C-reactive-protein-mediated protection of mice from Streptococcus pneumoniae.

Expression of a human C-reactive protein (CRP) transgene has been shown to protect mice from lethal Streptococcus pneumoniae infection. In the present study, we used cobra venom factor-induced decomplementation to investigate the role of complement in this CRP-mediated protection. An intact complement system significantly reduced pneumococcal bacteremia at 24 h postinfection and extended median survival time of both CRP-transgenic and nontransgenic mice. However, mortality was significantly lowered only for CRP transgenic mice. The transgene significantly reduced bacteremia for both normocomplementemic and decomplemented mice, but it resulted in a significantly longer median survival time and lower mortality only for normocomplementemic mice. These data suggest that in vivo complement and CRP amplify each other's protective capacity, particularly during the early course of infection.

Animals↗
PubMed1995

Crystal structure of a complement factor D mutant expressing enhanced catalytic activity.

Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195. These results provide a partial structural explanation for the low reactivity of "resting-state" factor D toward synthetic substrates.

Amino Acid Sequence↗
PubMed1995

Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice.

C-reactive protein (CRP) is an acute phase protein with a well known association with infection and other inflammatory conditions. Studies with use of purified CRP in in vitro assays provided early evidence that CRP has antibacterial activity. Subsequently it was shown that passively administered human CRP can protect mice from lethal infection with Streptococcus pneumoniae. In this study, we extend these observations to an in vivo model of host resistance by using human CRP transgenic mice. CRP transgenic mice experimentally infected with S. pneumoniae lived longer and had significantly lower mortality than their nontransgenic littermates. This increased resistance to infection was associated with q 10- to 400-fold reduction of bacteremia. Furthermore, male transgenics exhibited longer survival time than females, and this difference could be attributed to increased expression of CRP by males, which was mediated by testosterone. This study provides the first unequivocal evidence that CRP plays an important role in vivo in host defense against pneumococcal infections, and shows that sex hormones can affect expression of the human CRP transgene in mice.

Animals↗