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Biomedical subjects

W M Nauseef

Publications and source records attributed to W M Nauseef.

At least 55 records · Page 3Linked to original sources
PubMed1992

Roles of heme insertion and the mannose-6-phosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase.

Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme-free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO-related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Brefeldin A↗
PubMed1991

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.

Alkaloids↗
PubMed1991

A cytosolic inhibitor of human neutrophil elastase and cathepsin G.

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is alpha 1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase or 125I-cathepsin G with neutrophil cytosol or alpha 1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of alpha 1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100 degrees C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37 degrees C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either alpha 1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.

Ammonium Hydroxide↗
PubMed1991

Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558.

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.

Biological Transport↗
PubMed1990

Immunochemical and electrophoretic analyses of phosphorylated native and recombinant neutrophil oxidase component p47-phox.

Human polymorphonuclear neutrophils (PMNs) possess a potent oxygen-dependent microbicidal system that depends on the activity of a stimulus-activated multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Patients with chronic granulomatous disease (CGD) lack activity of this oxidase and consequently suffer severe and frequent infections. Components of the oxidase include both membrane-bound factors (most notably, cytochrome b559, which is absent in the X-linked form of CGD) and at least two cytosolic factors, one or the other of which is absent in autosomal CGD. Patients with CGD, particularly the autosomal type, have defective phosphorylation of proteins in the 44 to 48 Kd range. A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase system. Two-dimensional gel electrophoresis showed the identity of the 47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd protein that is phosphorylated in normal but not p47-phox-deficient CGD cytosol during activation of the NADPH-dependent oxidase. All full-length and C-terminal recombinant p47-phox proteins augmented the superoxide-generating capacity of the cell-free system and were phosphorylated when added to cytosol from normal subjects or from a patient with p47-deficient autosomal CGD. These studies provide compelling evidence that the 47-Kd cationic protein that is a substrate for phosphorylation during the activation of PMNs is, in fact, p47-phox, a cytosolic protein previously shown to be critical for normal activity of the NADPH-dependent oxidase of PMNs.

Autoradiography↗
PubMed1990

Two cytosolic components of the neutrophil NADPH oxidase, P47-phox and P67-phox, are not flavoproteins.

Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.

Arachidonic Acid↗
PubMed1990

Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src.

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.

Amino Acid Sequence↗
PubMed1990

Group B streptococcal polyarthritis complicating hemophilia B.

We report a case of polyarticular group B streptococcal infection in an HIV-negative 46-year-old alcoholic with factor IX deficiency. Septic arthritis occurs infrequently in the hemophilic population despite their chronic joint disease; indications for diagnostic arthrocentesis in these individuals are discussed. The group B streptococcus often behaves as an opportunist in adults.

Alcoholism↗
PubMed1990

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.

Biological Transport↗
PubMed1989

Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils.

Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.

Actin Cytoskeleton↗
PubMed1989

Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system.

Chronic granulomatous disease, a syndrome of recurrent infections and failure of oxidative microbicidal activity in phagocytes, results from defects in the gene for one of several components of an oxidase system that can undergo activation. To determine the relative prevalence of certain of the genetic variants of this disorder, we used immunoblotting to detect two specific neutrophil cytosolic proteins of 47 and 67 kd recently shown to be required for oxidase activation. Chronic granulomatous disease is usually an X-linked disorder associated with the absence of membrane cytochrome b558. Of our 94 patients with chronic granulomatous disease, however, 36 had a phenotype characterized by autosomal inheritance, normal membrane oxidase components (including cytochrome b558), and functionally defective cytosolic activity in a cell-free oxidase system. We studied 25 of these 36 patients and found that 22 lacked the 47-kd cytosolic protein, and the remaining 3 were missing the 67-kd component. Patients with chronic granulomatous disease whose functional defect was localized to the neutrophil membrane (classic X-linked cytochrome b-negative type and two other rare variants) had normal amounts of both cytosolic components. We estimate that approximately 33 percent of all patients with chronic granulomatous disease are missing the 47-kd cytosolic oxidase component and about 5 percent of patients are missing the 67-kd component. Chronic granulomatous disease caused by a defect in any cytosolic factors other than the 47-kd and 67-kd proteins, if it exists, is apparently rare.

Adolescent↗
PubMed1989

Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin.

Neutrophil guanine nucleotide-binding proteins are important components of receptor-mediated cellular responses such as degranulation, chemotaxis, and superoxide production. Because the cytoplasmic granules of neutrophils serve as an intracellular store of receptors and NADPH oxidase components, we investigated the subcellular distribution of substrates for ADP-ribosylation by both pertussis and cholera toxins. Cholera toxin substrates of Mr 43 and 52 kDa were present only in the plasma membrane fraction. A 39-kDa pertussis toxin substrate was present in the plasma membrane, cytosol, and a specific granule-enriched fraction. There were no substrates for either toxin in the primary granules. Quantitative GTP-gamma-5 binding was localized predominantly to the plasma membrane fraction (47%), but significant portions were found in the specific granule-enriched fractions (13%) and cytosol (34%) as well. Two-dimensional gel electrophoresis and chymotryptic digests of the pertussis toxin substrate from these three subcellular fractions suggested that they are highly homologous. Triton X-114 phase partitioning was used to investigate the hydrophobicity of the toxin substrates. The pertussis toxin substrates in the plasma membrane and granule fractions behaved like integral membrane proteins, whereas the cytosolic substrate partitioned into both lipophilic and aqueous fractions. ADP-ribosylation converted the substrates to a somewhat less lipophilic form. These data suggest that the specific granules or an organelle of similar density serve as an intracellular store of a G protein with a 39-kDa alpha-subunit and that the cytosolic fraction of neutrophils contains free alpha-subunits of the same size.

Adenosine Diphosphate Ribose↗
PubMed1989

Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.

Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.

Amino Acid Sequence↗
PubMed1989

Aberrant restriction endonuclease digests of DNA from subjects with hereditary myeloperoxidase deficiency.

Myeloperoxidase (MPO) is a critical component in the oxygen-dependent microbicidal activity of neutrophils. Hereditary deficiency of MPO occurs commonly, but its genetic basis has not been determined. Previously we have reported the presence of an 89-kilodalton protein, likely pro-MPO, in normal and MPO-deficient neutrophils and hypothesized that the absence of peroxidase activity in neutrophils from affected subjects was the result of defective posttranslational processing of pro-MPO. In this study we analyzed nucleic acids from three completely and two partially MPO-deficient individuals by using a cDNA probe for MPO. The affected individuals studied are unrelated to one another. Neutrophils from all affected subjects lacked mature MPO subunits; however, a monospecific antibody for MPO identified in these cells a high-molecular weight protein that is the same size as pro-MPO. Northern blots demonstrated that the amount and size of RNA (3.3 kilobases [kb]) in a completely deficient subject was normal. BglII digests of genomic DNA from control individuals (n = 14) contained three fragments that hybridized with cDNA for MPO under very stringent conditions. In contrast, BglII digests of genomic DNA from completely MPO-deficient individuals contained an extra fragment of 2.1 kb that was not present in DNA from controls. In addition, two different endonuclease digest patterns were found in MPO-deficient subjects who were biochemically and phenotypically identical. We conclude from these studies that (a) hereditary MPO deficiency is not associated with a major deletion or rearrangement of the MPO gene; (b) myeloid precursors in an MPO-deficient individual contain normal amounts of an mRNA that is the same size as that for MPO in normal individuals; and (c) the genetic basis for MPO deficiency may be heterogeneous, with at least two genotypes generating the same phenotype. These findings are consistent with the hypothesis that the genetic defect in MPO deficiency results in synthesis of a modified pro-MPO that undergoes defective posttranslational processing.

Blotting, Northern↗
PubMed1988

Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease.

Neutrophils kill microorganisms with oxygen radicals generated by an oxidase that uses the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as substrate. This system requires both membrane and cytosolic components and is defective in patients with chronic granulomatous disease. A cytosolic complex capable of activating latent membrane oxidase was eluted from guanosine triphosphate-agarose and was used to raise polyclonal antiserum that recognized 47- and 67-kilodalton proteins. These proteins were restricted to the cytosol of myeloid cells. Both proteins were associated with NADPH oxidase-activating capacity when neutrophil cytosol was purified on nucleotide affinity matrices or molecular sizing columns. Neutrophils from patients with two different forms of autosomal chronic granulomatous disease lacked either the 47- or 67-kilodalton protein.

Blotting, Western↗