Thesis (Ph. D.)--University of Hong Kong, 2005.

A study of the proteolytic enzymes of the sea anemone Metridium senile was undertaken in search of the proposed archetype 'serine protease' from which the family of homologous serine proteases of higher animals is believed to have arisen. Activity towards the chymotrypsin specific substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to be initially very low in homogenates of gastric filaments of Metridium but during incubation at 0°C an autocatalytic increase in the activity was observed. This 'activation' was inhibited by a competitive inhibitor of trypsin, benzamidine and by di-isopropylphosphorofluoridate (DFP) but not by the chymotrypsin competitive inhibitor indole. These observations suggested a process somewhat similar to the activation of serine proteases of higher animals. General steps in the purification of the anemone proteases were as follows: (i) 2 M LiCl extraction of acetone powder of gastric filaments (ii) acetone fractionation of the 2 M LiCl extract at -15°C, (iii) DEAE-Sephadex chromatography of the 40-80% acetone fraction, (iv) cation exchange chromatography of the DEAE-breakthrough peak on Bio-Rex 70 at pH 6.0 in the presence of .005 M indole and (v) re-chromatography on Bio-Rex 70 at pH 6.0 in the presence of indole. ATEE-ase activity was resolved into 3 components, designated A, BI and B2 in step (iv). Peaks A and BI migrated as single major Amido Black staining bands at pH 4.0 on polyacrylamide disc electrophoresis but there were several minor components present in both cases. Protease B2 appeared homogeneous on electrophoresis. 8 M urea-starch gel electrophoresis 32 of Dl³² P-labelled proteases followed by autoradiography; and Amido Black staining showed that the major Amido Black staining band was also the major radioactive band in each case. Gel filtration of protease A on G-100 Sephadex gave an approximate molecular weight of 40-50, 000. Sedimentation velocity of the enzymes gave values of S₂₀ of 2.0, 2.5. and 2.2 for DIP-proteases A, BI and B2, respectively, indicative of a lower molecular weight. Sedimentation equilibrium studies with protease A indicated considerable polydispersity but gave a weight-average molecular weight (Mw) of around 28, 000. The behavior on Sephadex G-100 and evidence of polydispersity upon sedimentation equilibrium may be a result of dimerization. Features of the amino acid composition showing similarity to α-chymotrypsin were a high half-cystine content and a similar number of aromatic and basic amino acids. Major differences from mammalian α-chymotrypsin included a higher proline and histidine content and a lower content of hydroxylic amino acids. Enzymic properties of the anemone enzymes were very similar to those of mammalian α-chymotrypsin. Protease A showed 3-5 fold higher k[subscript: cat] values in the hydrolysis of several chymotrypsin ester substrates but a markedly lower k[subscript: cat] in the hydrolysis of N-acetyl-L- tryptophan ethyl ester. The anemone enzyme was shown to be rapidly inhibited by DFP, phenyl methane sulphonyl fluoride (PMSF) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) but was not inhibited by the trypsin specific inhibitor N-[symbol omitted]tosyl-L-lysine chloromethylketone (TLCK). Competitive inhibition of the hydrolysis of ATEE was observed with indole and 3-phenyl propionate. Protease A hydrolysed the polypeptide chain of glucagon on the carboxyl side of tyrosine, phenylalanine and leucine, provided the latter was not adjacent to tyrosine or phenylalanine. A lower but significant cleavage at both sides of the two arginine residues was also observed. The latter cleavages were not affected by the chymotrypsin inhibitor TPCK but were eliminated by prior reaction of the enzyme with soybean trypsin inhibitor. Thus it was concluded that they represent the activity of a contaminating trypsin-like enzyme in the preparation of protease A. In studies on the short active centre sequence of proteased A, BI and B2 using the technique of co-electrophoresis of partial acid hydrolysates of Dl³² P-labelled enzymes and Dl³² P-chymotrypsin followed by high voltage electrophoresis and autoradiography, the active centre sequence of the three anemone proteases was found to be -Asp. Ser. Gly-, the sequence common to all of the mammalian serine proteases. In conclusion, the anemone enzymes show remarkable structural and enzymic similarity with mammalian chymotrypsin and they probably represent present day relics of early forms of chymotrypsin, homologous with the familiar enzyme of higher animals. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.

No description available.

Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

Thesis (Ph. D.)--University of Hong Kong, 1982.

Background and Objectives: Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells. The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D. Key findings: 1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples; 2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients; 3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis; 4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D. Conclusions: Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy