Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily.

Micronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A-domains) of blood coagulation factor XI (FXI) and plasma pre-kallikrein (PK). We have used mass spectrometry on a recombinant-expressed, putative A-domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A-domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A-domains and are therefore novel members of the PAN module superfamily, which also includes the N-terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A-domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein-protein or protein-carbohydrate interactions during parasite attachment and host cell invasion.

Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei.

The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.

Application of rapid read-out cleaning indicators for improved process control in hospital sterile services departments.

BACKGROUND: Heightened awareness of the importance of cleaning has led to an emphasis on automated systems for the decontamination of re-usable medical devices. The authors have previously described an enzymatic indicator system, based on thermostable adenylate kinases (tAK), for quantitative monitoring of automated cleaning processes within hospital sterile services departments (SSDs). AIM: To evaluate tAK indicators for routine process monitoring across a range of SSDs with different cleaning chemistries and different automated washer disinfectors (AWDs). METHODS: tAK indicator devices and alternative industry test indicators were included in five independent cleaning cycles in each of eight different AWDs. Residual tAK post wash was determined by a coupled luciferase assay using a modified hygiene monitoring system. FINDINGS: In all cases, with the exception of a single test, the alternative indicators showed that cleaning had been adequate. They were not able to discriminate between the performance of different processes. In contrast, the tAK indicators were able to resolve differences in the performance of processes across the different SSDs. Where the tAK indicators identified cleaning to the limits of detection of the assay, this demonstrated a log10 enzyme removal factor of >5.69. CONCLUSION: The results suggest that tAK indicators are suitable for providing improved process control for automated cleaning processes, being able to distinguish between wash performance in different hospital settings and between individual process runs. This technology is believed to be a useful addition to routine AWD performance qualification when used as a daily or weekly test.

Crystallization and preliminary X-ray analysis of active site-inhibited human coagulation factor VIIa (des-Gla).

Human coagulation factor VIIai that lacks the Gla domain (residues 1-44) has been prepared, purified, and crystallised. First, recombinant factor VII was activated to form factor VIIa, the active site was then inhibited with 1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone, and finally the Gla domain was removed by chymotryptic digestion, yielding factor VIIai (des-Gla). After further purification single crystals suitable for x-ray analysis were obtained by vapour diffusion. Crystals of factor VIIai (des-Gla) belong to the tetragonal space group P41212 or P43212 with unit cell dimensions a = b = 94.85 A, c = 114.30 A, contain one molecule per asymmetric unit, and diffract to 2.3-A resolution when exposed to synchrotron radiation.

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin.

The loop exchange mutant chymosin 155-164 rhizopuspepsin was expressed in Trichoderma reesei and exported into the medium to yield a correctly folded and active product. The biochemical characterization and crystal structure determination at 2.5 A resolution confirm that the mutant enzyme adopts a native fold. However, the conformation of the mutated loop is unlike that in native rhizopuspepsin and involves the chelation of a water molecule in the loop. Kinetic analysis using two synthetic peptide substrates (six and 15 residues long) and the natural substrate, milk, revealed a reduction in the activity of the mutant enzyme with respect to the native when acting on both the long peptide substrate and milk. This may be a consequence of the different charge distribution of the mutated loop, its increased size and/or its different conformation.

Expression and characterization of the human endothelin-A-receptor in Pichia pastoris: influence of N-terminal epitope tags.

Knowledge of the three-dimensional structure of the endothelin-A receptor (ET(A)) will provide important information for rational drug design of antagonists and agonists. In order to produce correctly folded and active receptor protein for physical characterization by such techniques as X-ray crystallography and circular dichroism spectroscopy, we have cloned and expressed the human ET(A)-receptor in Pichia pastoris. The expression constructs all contained signal sequences to direct the expressed protein to the membrane fraction. Fidelity of folding and the subtype specificity of the expressed receptor was demonstrated by ligand binding studies using 125I-labelled endothelin-1 (ET-1). Expression of receptor with two different N-terminal epitope 'tags' had little effect on the affinity of ET-1 for the receptor. The strain of P. pastoris employed did influence the quantity of receptor expressed.

Biochemical studies on a case of feline mannosidosis.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.