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P J Tatnell

Publications and source records attributed to P J Tatnell.

14 recordsLinked to original sources
PubMed1998

Mouse procathepsin E gene: molecular organisation and chromosomal localisation.

A 15.6 kb genomic clone encompassing the mouse procathepsin E gene was isolated and mapped. Sequencing revealed that the gene consists of nine exons followed by a polyadenylation signal at the 3'-end. The 5'-flanking region appears to be a TATA-less promoter but contains a nucleotide sequence that matches perfectly with the consensus motif of an initiator element [S.T. Smale, Biochim. Biophys. Acta 1351 (1997) 73-88.] to direct accurate initiation of transcription by RNA polymerase. This overlaps the site that was determined for the start of transcription. The absence of features considered typical of TATA-box regulated or housekeeping types of genes is consistent with the low levels of procathepsin E gene expression that are normally observed and might imply a unique sensitivity to or requirement for tissue-specific transcription factors that would account for the sporadic distribution of this aspartic proteinase in cells and tissues. The single copy of the procathepsin E gene was located on chromosome 1, near to that of mouse prorenin, a closely related aspartic proteinase involved in blood pressure regulation.

Amino Acid Sequence↗
PubMed1997

Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation.

Cathepsin E is an aspartic proteinase that has been implicated frequently in Ag processing for presentation on class II MHC molecules, but no information exists on its cleavage specificity within Ags in relation to known T cell epitopes. We have analyzed the processing by cathepsin E of a large C-terminal domain of tetanus toxin (residues 872-1315), and we have compared the processing products with those liberated by cathepsin D, a related aspartic proteinase also thought to be involved in class II MHC-restricted Ag processing. Processing products were analyzed by N-terminal Edman degradation and mass spectrometry following reverse-phase HPLC separation of peptides. A total of 28 cleavage sites was identified, 11 of which were recognized by both cathepsins E and D. Most, although not all, sites were between pairs of hydrophobic residues and were located within the 200-amino-acid C terminal region known to contain several human T cell epitopes. Previously described T cell epitopes, for example, between residues 1273 and 1284, were flanked by cathepsin E and D cleavage sites. These data are consistent with an important role for cathepsins E and/or D in Ag processing in the human immune system.

Amino Acid Sequence↗
PubMed1997

Cloning, expression and characterisation of murine procathepsin E.

The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli. The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors. The majority of the recombinant enzyme was present as a homodimer (Mr approximately 80) in which the two monomers were linked by an intermolecular disulfide bond. By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase. The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate 'knock-out' mice deficient in this important aspartic proteinase to be undertaken.

Amino Acid Sequence↗
PubMed1995

Monomeric human cathepsin E.

Cathepsin E is a homodimer, consisting of two monomers linked by an inter-molecular disulphide bond. The cysteine residue involved is located near to the N-terminus of the mature proteinase. By mutating this residue to alanine, a monomeric form of human cathepsin E was engineered and purified. The activity of the resultant enzyme was not altered significantly (in terms of its ability to hydrolyse two chromogenic peptide substrates; and its susceptibility to inhibition by pepstatin). However, the stability of the mutant enzyme to alkaline pH and to temperature was markedly reduced.

Amino Acid Sequence↗
PubMed1994

GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa: evidence from metabolite studies.

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

Alginates↗
PubMed1993

A metabolic study of the activity of GDP-mannose dehydrogenase and concentrations of activated intermediates of alginate biosynthesis in Pseudomonas aeruginosa.

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

Alginates↗
PubMed1992

Isolation of a mucoid alginate-producing Pseudomonas aeruginosa strain from the equine guttural pouch.

The isolation and characterization of a mucoid, alginate-producing strain of Pseudomonas aeruginosa from a nonhuman host, namely, in chondroids from an equine guttural pouch, is reported for the first time. Pure cultures of P. aeruginosa 12534 were isolated from a 17-month-old pony mare with a history of chronic bilateral mucopurulent nasal discharge from the right guttural pouch. Transmission electron microscopy of chondroids showed mucoid P. aeruginosa growing as microcolonies within a matrix of extracellular material. On the basis of expression of the mucoid phenotype under different growth conditions, P. aeruginosa 12534 belongs to group 1 and resembles other isolates carrying the muc-23 mutation. The bulk of the extracellular material was characterized as being alginate by chemical and 1H nuclear magnetic resonance analyses, which showed that it had a composition similar to that produced by isolates of P. aeruginosa from human patients with cystic fibrosis.

Alginates↗