Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants - identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

[Lymphohistiocytic activation syndrome and Burkholderia cepacia complex infection in a child revealing chronic granulomatous disease and chromosomal integration of the HHV-6 genome]. / Syndrome d'activation lymphohistiocytaire associé à une infection àBurkholderia cepacia complex chez un nourrisson révélant une granulomatose septique et une intégration génomique du virus HHV-6.

Chronic granulomatous disease (GCD) is characterized by severe infections, notably with Burkholderia cepacia complex (BCC). GCD is rarely complicated by lymphohistiocytic activation syndromes, most often secondary to bacterial or viral infections, in particular human herpes virus 6 (HHV-6). We describe the case of a 10-month-old boy who suffered from multiple organ failure due to a BCC infection and a lymphohistiocytic activation syndrome, leading to diagnosis of GCD. The initial search for HHV-6 was positive and the infection was treated, but the progression and viral sample analysis led to the chromosomal integration of the HHV-6 genome. The child's clinical condition was normal after bone marrow transplantation. This case describes a rare association between GCD and lymphohistiocytic activation syndrome and raises questions about the role played by chromosomal integration of the HHV-6 genome.

[Chronic-granulomatous disease]. / La granulomatose septique chronique.

Chronic-granulomatous disease (CGD) is a rare inherited primary immunodeficiency syndrome caused by a defective oxidative metabolism of phagocytic cells. Dysfunction of the membranous NADPH oxidase complex leads to a greatly increased susceptibility to severe fungal and bacterial infections, early in childhood. The most severe and frequent type of GCD is the X-linked transmitted form caused by mutations in the CYBB gene encoding the redox element of the oxidase complex, gp91phox or Nox2. However, very rare autosomal recessive CGD affecting other oxidase components than Nox2 are characterized by mild-clinical manifestations that could appear later at the adult age. Long-term antibiotic prophylaxis is essential to prevent infections associated with CGD, but approaches based on hematopoietic stem-cell transplantation and gene therapy offer valuable hope in a near future.

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.

The 23-kilodalton protein, a substrate of protein kinase C, in bovine neutrophil cytosol is a member of the S100 family.

A bovine neutrophil protein termed p23 because of an apparent molecular mass of 23 kDa in SDS-PAGE is present in large amounts both in a soluble form in the cytosolic fraction of bovine neutrophil homogenates and associated to the cytoskeleton. P23 is accompanied during the first steps of the purification procedure by a smaller size protein termed p7 on the basis of a rate of migration in SDS-PAGE corresponding to a 7-kDa protein [Stasia, M. J., Dianoux, A. C., & Vignais, P. V. (1989) Biochemistry 28, 9659-9667]. The two proteins, p23 and p7, have been purified to homogeneity by an improved procedure consisting of two chromatographic steps. The electrospray mass spectrometry technique applied to p23 and p7 indicated molecular masses close to 17 and 10 kDa, respectively, significantly different from the masses derived by SDS-PAGE. Bovine neutrophil p23 and p7 presented large primary structure homologies with two human proteins, MRP14 and MRP8, which are expressed in large amounts in macrophages under conditions of chronic inflammation. In addition, p23 and p7 cross-reacted with monoclonal antibodies specific of MRP14 and MRP8. Bovine p23 and p7 bound Ca2+, and their amino acid sequences contained two Ca(2+)-binding domains per protein, largely identical to those of human MRP14 and MRP8. Bovine p23 and p7 associated together to form a heterodimeric complex, which largely escaped attack by trypsin, whereas the isolated p23 and p7 components were readily digested. These features are typical of Ca(2+)-binding proteins belonging to the S100 family.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility.

A 24-kDa G protein, ADP-ribosylable by exoenzyme C3 from Clostridium botulinum and therefore related to the rho family, was found to be abundantly present in human and bovine neutrophils, and preferentially located in cytosol. In human myeloid HL60 cells, the amount of C3 substrate increased during differentiation of the HL60 cells into granulocytes. The effect of exoenzyme C3 on different functions of bovine neutrophils, namely generation of O-2, degranulation and chemotaxis, has been tested, using electropermeabilized cells. Exoenzyme C3 hardly affected the respiratory burst and the degranulation. In contrast, it efficiently inhibited the spontaneous and chemoattractant-induced motility of the cells and disorganized the actin microfilament assembly.

Immunocharacterization of beta- and zeta-subspecies of protein kinase C in bovine neutrophils.

The isoforms present in a crude preparation of bovine neutrophil protein kinase (PKC) were identified by immunodetection with antibodies directed against specific sequences of bovine and rat brain PKC isozymes. The major isoform of bovine neutrophil PKC was identified as beta-PKC and the minor one as zeta-PKC.

A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization.

In 32Pi-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. It was purified from bovine neutrophil cytosol by a series of chromatographic steps, including ion exchange on DE-52 cellulose and Mono Q, and filtration on Bio-Gel P60 in the presence of mercaptoethanol and urea. The apparent molecular mass of the purified protein, assessed by SDS-PAGE and mercaptoethanol by reference to protein markers, ranged between 20 and 23 kDa, depending on the percentage of polyacrylamide and conditions of migration. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Some properties of the 23-kDa protein, including its amino acid composition, were determined. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of four discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [gamma-32P]ATP in the presence of bovine neutrophil PKC supplemented with Ca2+, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. The apparent KM of ATP was 9 microM. The 23-kDa protein was also phosphorylated by PKM, the catalytic fragment of PKC obtained after removal of the regulatory domain, but not by cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of protein kinase C from polymorphonuclear neutrophils by long chain acyl coenzyme A and counteraction by Mg-ATP.

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) from bovine polymorphonuclear neutrophils was inhibited by micromolar amounts of long chain acyl-CoAs. The extent of inhibition at a given concentration of the acyl-CoAs depended on the length of the chain. A chain length of at least 12C was required for inhibition. Inhibition of protein kinase C activity was counteracted specifically by Mg-ATP.