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W J Griffiths

Publications and source records attributed to W J Griffiths.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterisation of variant forms of prophenin: mechanistic aspects of the fragmentation of proline-rich peptides.

Prophenin 1 (PF-1) is a 79-residue polypeptide originally isolated from porcine leukocytes. Its amino acid sequence has been determined by a combination of mass spectrometry and Edman degradation (Harwig SSL. et al. FEBS Lett. 1995; 362: 65). Prophenin (PF) and variants thereof are also found in organic extracts of porcine pulmonary tissue (Wang Y. et al. FEBS Lett. 1999; 460: 257). In the present study we have characterised the variant forms of PF found in these extracts using nano-electrospray (nano-ES) high resolution and tandem mass spectrometry. The major forms of PF found in these extracts by nano-ES mass spectrometry are the 80-residue polypeptides prophenin-2-Pyr (PF-2-Pyr) and prophenin-2-Gln (PF-2-Gln). Prophenin-2-Pyr is refractory to Edman degradation due to the presence of an N-terminal pyroglutamic residue. In PF-2-Gln the N-terminal residue is glutamine and the C-terminus is amidated. In porcine pulmonary extracts PF-1 is present to only a minor extent. Other shorter polypeptides are also found in these extracts including 18- and 17-residue C-terminal fragments of PF. The primary structure of PF is highly unusual in that it shows four almost perfect decamer repeats of FPPPN(V/F)PGPR and, out of the 79/80 residues, 42 are proline and 14 are phenylalanine. Tryptic digestion of PF gives peptides containing the decamer repeat and collision-induced dissociation of these peptides provides an insight into the fragmentation mechanisms of proline-rich peptides. Facile cleavage within the Pro-Pro-Pro sequence of these peptides suggests the involvement of a cyclic peptide in the fragmentation mechanism. Fragmentation mechanisms that account for the formation of fragment ions at other cleavage sites are also discussed.

Amino Acid Sequence↗
PubMed2000

Nanospray mass spectrometry in protein and peptide chemistry.

The introduction of electrospray in the mid-1980s revolutionised biological mass spectrometry, in particular in the field of protein and peptide sequence analysis. Electrospray is a concentration-dependent, rather than a mass-dependent process, and maximum sensitivity is achieved at low flow rates with high-concentration, low-volume samples. This has lead to the development of nanoelectrospray, microelectrospray and related low flow-rate forms of electrospray which offer high sensitivity with low sample consumption. In this chapter the physical chemistry of low flow-rate electrospray is discussed, and a brief review of the types of low flow-rate electrospray interfaces is made. An indication of the performance obtainable on various instruments is given, along with some comments from the author's own experience of these techniques.

Mass Spectrometry↗
PubMed2000

De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/collision-induced dissociation mass spectrometry.

A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.

Aldehyde Dehydrogenase↗
PubMed2000

Localization of iron transport and regulatory proteins in human cells.

The recent discovery of HFE, the MHC-Class-I-like gene mutated in up to 90% of patients with hereditary haemochromatosis, and the gene encoding the Nramp2/divalent metal transporter-1 (DMT-1) implicated in ferrous iron transport holds promise for a greater understanding of human iron metabolism. Since the HFE protein can be crystallized as a ternary complex with the transferrin receptor and iron-saturated transferrin, and DMT-1 expression is up-regulated in hereditary haemochromatosis, these proteins are likely to interact in a common pathway for human iron homeostasis. To investigate the cellular interactions between the cognate proteins encoded by these genes, we generated a panel of rabbit and avian antisera from human HFE and DMT-1 derived peptides. The antibodies were characterized by ELISA reactions and Western immunoblotting. Immunohistochemical staining showed that DMT-1 protein localized to the brush border of human duodenum where it is predicted to serve as the principal transporter of ferrous iron from the intestinal lumen. In the human cell lines, Caco-2 (small intestinal phenotype upon differentiation) and K562 (erythroleukaemic) HFE, in the presence of iron-saturated transferrin, co-localized with transferrin receptors in an early endosome compartment using confocal immunofluorescence microscopy. This interaction may be critical in small-intestinal crypt cells which express HFE, where it may function to modulate their intrinsic iron status thereby programming iron absorption by DMT-1 in the mature enterocyte. In undifferentiated Caco-2 cells, DMT-1 localized to a discrete late endosome compartment distinct from that occupied by HFE where, in addition to brush-border iron uptake, it may function to regulate the availability of iron delivery to intracellular iron pools. Disruption of the HFE gene as a result of mutations associated with hereditary haemochromatosis may thus impair homeostatic mechanisms controlling iron absorption within the small-intestine epithelium by a direct interaction with transferrin receptors and by subsequent alteration of DMT-1 expression. Identification of the molecular interactions of HFE with DMT-1 and other key components of the iron transport pathway has implications for a mechanistic understanding of the pathophysiology of human iron storage diseases as well as the regulation of normal iron balance.

Animals↗
PubMed2000

Possible release of an ArgGlyArgProGln pentapeptide with innate immunity properties from acidic proline-rich proteins by proteolytic activity in commensal streptococcus and actinomyces species.

This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral Streptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr(1)-Pro(104)Pro(105) and a Gly(111)-Pro(149)Gln(150) peptide together with a presumed Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) pentapeptide. The synthetic Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro.

Actinomyces↗
PubMed1999

Porcine pulmonary surfactant preparations contain the antibacterial peptide prophenin and a C-terminal 18-residue fragment thereof.

Surfactant preparations obtained from porcine lungs by extraction with chloroform/methanol followed by chromatography over Lipidex-5000 are used for treatment of respiratory distress syndrome in preterm infants. These preparations contain about 98% phospholipids and 1-2% of the hydrophobic pulmonary surfactant-associated proteins B and C (SP-B and SP-C). Separation of the proteins in the surfactant preparation by reversed-phase high performance liquid chromatography revealed, in addition to SP-B and SP-C, the presence of three peptides derived from the cathelicidin family of antibacterial peptides. The 79-residue proline-rich peptide prophenin (identical to that isolated from leukocytes), an 80-residue prophenin with an N-terminal pyroglutamic acid residue, and a C-terminal 18-residue fragment of prophenin were found in approximate molar ratios of 1:20:5. A synthetic version of the C-terminal 18-residue peptide exhibits salt-dependent antibacterial activity (higher activity in the absence of salt) against the Gram-positive bacterium Bacillus megaterium Bm11 and, to a lesser extent, against Gram-negative Escherichia coli D21 cells. It appears possible that the presence of prophenin peptides may contribute to the antibacterial properties of surfactant preparations.

Amino Acid Sequence↗
PubMed1999

Nano-electrospray tandem mass spectrometry for the analysis of neurosteroid sulphates.

Neurosteroids are synthesised in the central and peripheral nervous system or are derived from peripheral sources, and act in the nervous system. In the present study we have evaluated the potential for using nano-electrospray (nano-ES) tandem mass spectrometry (MS/MS) for the structural analysis and detection of neurosteroids, in particular, steroid sulphates found in brain. Complete structural information can be obtained from 1 ng (3 pmol) of steroid sulphate, while fragment ions characteristic of the sulphate ester group can be obtained from only 3 pg (10 fmol) of sample. These values correspond to the expected quantities of steroid sulphates (e.g. pregnenolone sulphate) in about 100 mg and 300 microg of brain, respectively. Deuterated neurosteroid sulphates added to homogenised rat brain have been successfully analysed by nano-ES-MS/MS at a level of 50 pg/mg of brain.

Animals↗
PubMed1999

Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time-of-flight tandem mass spectrometer.

The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.

Amino Acid Sequence↗
PubMed1999

Inherited disorders of iron storage and transport.

Diverse hereditary disorders associated with iron accumulation cause widespread organ damage. New insights into cellular pathways of iron transport have emerged from the identification of molecules implicated in heritable defects of iron metabolism. Unravelling the genetic basis of rare variants of haemochromatosis should provide vital functional information to further our mechanistic understanding of iron homeostasis.

Adult↗
PubMed1999

Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry.

The characterization of conjugated metabolites of benzo[a]pyrene (BP) in the urine of male germ-free rats given a single intraperitoneal dose of [(14)C]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pak C(18) cartridge and separated by lipophilic ion-exchange chromatography into neutral and acidic fractions (fractions I-V). Metabolites in the latter fractions, constituting more than 80% of the urinary radioactivity, were characterized by reversed-phase HPLC and capillary column liquid chromatography/electrospray mass spectrometry (LC/ESMS) and tandem mass spectrometry (MS/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity. Some coeluting compounds were semiquantified from the ion current chromatograms obtained in the capillary column LC/ESMS analyses. The major conjugated metabolites in fraction II, containing about 50% of the urinary radioactivity, consisted of three tetrahydrotrihydroxy-BP-S-N-acetylcysteines, the major isomer being 7,8,9,10-tetrahydro-8,9, 10-trihydroxy-BP-7-S-N-acetylcysteine, two dihydrotrihydroxy-BP-S-N-acetylcysteines, and a tetrahydrotetrahydroxy-BP-S-N-acetylcysteine. Fraction II also contained three apparently unconjugated compounds whose structures will be described elsewhere. Metabolites characterized in fractions III and IV, containing about 30% of the urinary radioactivity, included three BP-O,O'-disulfates, two monohydroxy-BP-O-sulfates, three dihydrodihydroxy-BP-O-sulfates, three BP-O,O'-diglucuronides, and a BP-O-sulfate-O'-glucuronide. Trace levels of a tetrahydrotrihydroxy-BP-S-glutathione conjugate were detected in fraction V.

Animals↗
PubMed1998

Dolichol-like lipids with stimulatory effect on DNA synthesis: substrates for protein dolichylation?

Substantial evidence has suggested that a nonsterol product of mevalonic acid (MVA) is essential for the initiation of DNA synthesis in mammalian cells. Several possible isoprenoid candidates have been suggested, but the identity of this compound still remains unknown. In this study we have isolated and purified MVA products from SV40-transformed human fibroblasts and identified fractions with a growth-stimulatory effect. The cells were labelled with [14C]MVA in the presence of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. After lipid extraction, the [14C]MVA-labelled lipids were subjected to high performance liquid chromatography and size-exclusion chromatography, and the effect of the fractionated eluate on the DNA synthesis of arrested MVA-depleted target cells was tested. Thereby we found a fraction of [14C]MVA-labelled lipids with a substantial stimulatory effect on DNA synthesis. The chromatographic behavior suggested that the growth-stimulating fractions contained dolichol-20. This was confirmed by mass spectrometric analysis. Similar results were obtained when lipids from hepatocellular carcinoma cells and a sample from breast tumor were isolated and analyzed by the same procedure. The mechanisms by which these compounds induce DNA synthesis are unknown. Recent data obtained in our laboratory have provided evidence that dolichyl groups are covalently linked to tumor cell proteins, which implicates a new biological function for long-chain polyisoprenoid alcohols (Hjertman et al. [1997] FEBS Lett 416:235-238). In this study we demonstrate that tumor cells containing dolichol-like growth-stimulatory lipids also contained dolichylated proteins. This raises the question whether the growth-stimulatory dolichol-like lipids serve as substrates for the dolichylation reaction.

Cell Division↗
PubMed1998

Synthetic peptide-containing surfactants--evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution.

Pulmonary surfactant contains two hydrophobic proteins, SP-B and SP-C. With the aim of identifying synthetic SP-B and SP-C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21-residue peptide KL4. This peptide, designated KL(2,3) from its spacing of nonpolar and polar residues, exhibited similar alpha-helical content as KL4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL4 in the same environment. The second amphipathic peptide analyzed was succinyl-LLEKLLEWLK-amide (WMAP10). KL4 more efficiently accelerated the spreading of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Pam2GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68:22:9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL4, but not KL(2,3), when present in an interfacial monolayer composed of Pam2GroPCho/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 7:3 (by mass), increased lipid insertion from subphase vesicles. An SP-C analogue, SP-C(Leu), with all helical valyl residues in native SP-C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP-C(Leu) exhibited similar alpha-helical content as native SP-C and a transmembrane orientation and, in contrast to poly-valyl-containing synthetic peptides, it folds into a helical conformation after acid-induced denaturation. SP-C(Leu) accelerated the spreading of Pam2GroPCho/PtdGro/PamOH, 68:22:9 (by mass), almost identically to native SP-C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2% (by mass) SP-C(Leu) combined with Pam2GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30% compared with untreated controls.

Animals↗
PubMed1998

Demonstration of leukotriene-C4 synthase in platelets and species distribution of the enzyme activity.

Human platelets have been demonstrated to possess leukotriene (LT)-C4 synthase activity and may thus be involved in transcellular 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4) synthesis. In this study, platelets from seven different species were screened for LTC4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70% conversion of 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA4) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC4 after incubation with LTA4. The results demonstrate that LTC4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), indicating the presence of LTA4 hydrolase in porcine platelets. A protein with a molecular mass of approximately 18 kDa and LTC4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N-terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed Km (for LTA4) and Vmax values of 3.3 microM and 521 nmol x mg protein(-1) x min(-1), respectively. In addition, the presence of a mRNA transcript encoding LTC4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N-terminus demonstrated very high identity between equine and human leukotriene-C4 synthase in this region. In summary, the present study establishes that platelets contain LTC4 synthase and indicates that this enzyme is widely distributed among platelets from various species.

Amino Acid Sequence↗
PubMed1998

Analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene found in rat urine by nano-electrospray mass spectrometry.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

Acetylcysteine↗
PubMed1998

Analysis of variant forms of porcine surfactant polypeptide-C by nano-electrospray mass spectrometry.

Electrospray (ES) mass spectrometry has been used to analyse preparations of porcine pulmonary surfactant polypeptide-C (SP-C). A number of variant forms of the native 35-residue dipalmitoylated peptide were detected including (a) C-terminally methylated SP-C, (b) C-terminally methylated and methionine oxidized SP-C, (c) N-terminally truncated, C-terminally methylated and methionine oxidized SP-c, (d) C-terminally elongated, C-terminally methylated and methionine oxidized SP-C, and (e) tripalmitoylated, C-terminally methylated and methionine oxidized SP-C. C-terminal methylation and methionine oxidation are probably a consequence of the sample handling procedure. The occurrence of the C-terminally elongated form of SP-C has implications for the in vivo processing of proSP-C and the Tandem mass spectrometry (MS/MS) was used to confirm the amino acid sequence of SP-C and the presence of palmitoyl groups covalently linked to the peptide. Some of the structures of the variant forms of SP-C were determined by MS/MS.

Amino Acid Sequence↗
PubMed1997

Evidence for protein dolichylation.

Labeling of human colon carcinoma cells with [3H]dol, followed by extensive delipidation and removal of dol-P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and > 140 kDa. HPLC purification of proteolytic products of [3H]dol- and [35S]cys-labeled proteins revealed a hydrophobic peak containing both dol and cysteine. The dol/cys-labeled products were clearly separated from GG-cys, and exhibited a hydrophobicity between that of dol-P and dol. In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds. After HPLC purification of released dol-like lipids, these were subjected to mass spectrometry. This demonstrated molecular ions with the same mass as that of dol. Taken together our data provide evidence for the existence of proteins covalently modified with dol.

Autoradiography↗
PubMed1997

High levels of (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide in the serum and urine of children with severe cholestatic liver disease.

Extracts of urine and serum from children with cholestatic liver disease were analyzed by fast atom bombardment (FAB) mass spectrometry. About half of all spectra showed a peak at m/z 657, compatible with the presence of a glucuronidated cholestenediol sulfate. Separation by ion exchange chromatography before and after solvolysis and treatment with beta-glucuronidase, combined with analyses by gas chromatography-mass spectrometry and FAB mass spectrometry with collision-induced dissociation, showed that the major compound responsible for the peak at m/z 657 was (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide. The double conjugate of 27-hydroxycholesterol was also identified and double conjugates of cholestene- and cholestanetriols were also present. Semiquantitative analyses of the double conjugate of 24-hydroxycholesterol in patients whose FAB spectra showed a peak at m/z 657 indicated serum levels of 2-18 microM and a daily urinary excretion of 0.1-2.7 mumol/24 h. Eleven of 13 studied patients with a prominent peak at m/z 657 in the FAB spectra of their serum or urine either underwent liver transplantation or died. It is concluded that double conjugation of hydroxysterols with sulfuric and glucuronic acids can be an important metabolic pathway, particularly for (24S)-24-hydroxycholesterol. It is speculated that serious cholestatic liver disease may induce an increased formation and release of (24S)-24-hydroxycholesterol from brain (Lütjohann et al. 1996, Proc. Nutl. Acad. Sci. USA. 93: 9799-9804) with subsequent extracerebral conjugation with sulfuric and glucuronic acids.

Biomarkers↗