Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, 2007. / "June 2007" Includes author's previously published papers. Bibliography: leaves 187-208. Also available in print form.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 162-175).

Thesis (Ph.D.)--Ohio University, March, 2006. / Includes bibliographical references (leaves 105-111)

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Human Cytomegalovirus (HCMV) is the most common cause of viral congenital infection in the world. Around 5-10% of HCMV infected children are symptomatic at birth, and 50-90% of these develop severe manifestations with a 30% mortality rate. Among the asymptomatic children at birth, 10-15% develop late sequelae. The major cell entry glycoproteins of HCMV form three complexes: gC-I containing gB; gC-II containing gM &amp; gN; and gC-III containing gH, gL, and gO (or UL128-131). These entry glycoproteins are polymorphic, producing different glycoprotein genotypes. The polymorphic nature of the glycoproteins as well as their ability to elicit neutralizing antibodies made them of interest in correlating them with the severity and outcome of the disease. This study aimed to develop a robust system to identify clusters of glycoprotein genotypes and to correlate them with disease manifestation. PCR assays of high sensitivity were used to identify all six glycoproteins. The PCR products were digested using restriction enzymes (RFLP) to identify the glycoprotein genotypes. Available laboratory strains (AD169, Towne, Davis, Toledo, and Merlin) as well as 112 clinical samples were amplified and genotyped using the assay, and their glycoprotein genotype profile was determined. A reliable sensitive assay was successfully developed to identify all glycoprotein genotypes including a novel gM assay using PCR/RFLP. The clinical samples were grouped according to disease manifestation (Group 1: congenital/postnatal patients (subgroup 1A: confirmed congenital patients &amp; subgroup 1B: patients with either congenital or postnatal infection), Group 2: immunocompetent patients, Group 3: immunocompromised patients (subgroup 3A: immunocompromised patients with primary infection, subgroup 3B: immunocompromised patients with recurrent infection &amp; subgroup 3C: immunocompromised patients with unconfirmed primary or recurrent infection)). Genotype gB1 was found predominantly prevalent in congenital/postnatal and immunocompromised patients, while gB3 was the most common genotype in immunocompetent patients. This result along with the phylogenetic analysis performed in this study suggest a relationship between gB genotypes and the immune response of the patients, where gB3 may be positively selected by host immune pressure. The novel gM assay genotyped the highly conserved gene (UL100) into three distinct genotypes; gM3 genotype associated with the congenital/postnatal group; which may provide an insight into understanding viral attachment and spread into the host cell. In congenital/postnatal infection, gH1 (72.7%) and gL4 (65.1%) were the most prevalent genotypes (gH1= 32/44, gL4= 28/43; P=0.000). In immunocompetent patients, mixed gH and mixed gL genotypes significantly correlated with the group, and in the immunocompromised group gH2 and mixed gL genotype were the most common genotypes (51.1% and 46.9% respectively). Glycoproteins gO, gH and gL are components of gC-III complex and gO1 was found to be the most prevalent gO genotype in all infection types (Group 1= 32.1%, Group 2= 85.7%, Group 3= 18.8%; P < 0.05). Also, in congenital/postnatal infection gN and gO were found to significantly link with each other and this is expected since both glycoproteins are highly polymorphic and are located on adjacent gene loci in HCMV genome (gN1+gO1a (P=0.000), gN3a+gO4 (P= 0.000)). The specific gN-gO linkages found here could be potential indicators for congenital/postnatal infection. In congenital/postnatal infection group, gH had significant linkages to gN and gO (gH1+gN1 (P=0.023, gH1+gO1a (P=0.013)) suggesting that interlinked selection of glycoprotein genotypes in the gC-II and gC-III complexes is involved in the development of congenital infection. High viral loads were found trending with immunocompromised patients, while low viral loads were significantly associated with mixed infected patients. This study has shown significant associations between a number of glycoproteins and congenital infection. Previously ignored glycoproteins gM and gL have been shown to be potentially of significant interest in this study and a larger study to confirm this is needed. In most cases the pattern of glycoprotein genotypes in congenital infection is more similar to that of immunocompromised than immunocompetent patients and it is possible that immune pressure is selecting for or against particular glycoprotein genotypes. The relationship between mixed infection and sample type may offer opportunities for development of prognostic biomarkers for congenital disease and further work is warranted.

A structural glycoprotein has been extracted from bovine ligamentum nuchae, using 5M guanidine hydrochloride containing a disulphide bond reducing agent, and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse with a molecular weight of ~ 34 OOO as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetyl-glucosamine, galactose and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity using 3H lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2h incubation was ~ 60 x 10⁴ dpm/mg purified protein. Addition of 10mM lysine resulted in an ~ 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using 3H lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27mM aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by ~ 14%. In the absence of 5M guanidine hydrochloride and a reducing agent, the glycoprotein undergoes aggregation which in the presence of copper ions results in the formation of cylindrical tactoids, the diameter of which (11nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibres.

The work presented within this thesis expands upon the theme within this laboratory, of utilising epitope-labelled recombinant proteins for the construction of multivalent subunit vaccines. Mammalian-cell expression- vectors were constructed which encoded a 14 amino acid epitope-tag, termed Pk-tag. The genes encoding the haemagglutinin (PIN) and fusion (F) glycoproteins (model type II and type I proteins respectively) from the paramyxovirus simian virus 5 (SV5), were inserted into the above vectors such that the sequence encoding the Pk-tag was present at the amino (N) or carboxy (C) terminus of SV5 HN, and the C-terminus of SV5 F. The genes were expressed in mammalian cells by utilising the vaccinia virus/T7 transient-expression system. Encouraging results were obtained which demonstrated that the addition of the Pk-tag to the N or C termini of SV5 HN, or to the C-terminus of SV5 F, did not prevent the production of; full length, N-linked glycosylated, oligomeric, natively folded and cell-surface localised Pk-tagged protein. An attempt was made to produce secretable forms of Pk-tagged SV5 HN and F. For this purpose, a vector was constructed which encoded a truncated version of the SV5 F protein in which the C-terminal transmembrane anchor & cytoplasmic tail were deleted, but which still possessed the sequence encoding the C-terminal Pk-tag at the C-terminus of the ectodomain. Expression of this gene in mammalian cells resulted in the production of a protein which had undergone N-linked glycosylation and partial oligomerisation. No secretion of the truncated Pk-tagged protein into the external milieu was detected. Furthermore, production of potentially secretable forms of N & C- terminally Pk-tagged SV5 HN was achieved by the construction of plasmid vectors in which the non-cleavable native HN signal sequence was replaced by a putative cleavable signal sequence from the Epstein Barr virus gp220/360 glycoprotein. Expression of the modified Pk-tagged HN genes in mammalian cells produced proteins of a lower mwt. than expected and which, apart from a small proportion of the N-terminally Pk-tagged molecules, did not possess N-linked oligosaccharides and were not recognised by conformationally sensitive mAbs. No secretion of the modified Pk-tagged HN into the external milieu was detected. Following the first initial characterisation of the Pk-tagged HN & F, in which very encouraging results were obtained, an attempt was made to isolate cell-lines which constitutively or inducibly expressed Pk-tagged HN. Production of Pk-tagged HN could not be detected from constructs in which expression was driven from constitutive promoters. However, production of N-terminally Pk-tagged HN (but not C-terminally tagged HN) was detected when expression was driven by the tTa inducible expression system. As a further development to the tTa system, a 293 cell-line was isolated which expressed high-levels of functional tTa. The tTa-producing cell-line was subsequently utilised in an attempt to isolate cell-lines which inducibly produce N-terminally Pk-tagged HN. These cells are currently undergoing selection for drug resistance. Further experiments were performed to try and develop a system whereby the low copy number of episomally-maintained EBV-based vectors present in a stable cell-line could be amplified to high copy number, whereby a subsequent increase in protein production would be envisaged. For this purpose, SV40 ori-containing, EBV-based vectors were constructed in which the expression of the non-toxic SV5 P protein was under the control of the hCMV IE promoter/enhancer. Cell-lines were isolated which produced the SV5 P in various amounts. Transient amplification of the episomal copy number was attempted via a transient expression of the SV40 LTAg, using plasmid DNA transfection. No subsequent increase in protein production was observed by way of a Western blot analysis.

The secreted acid phosphatase (SAcP) of Leishmania donovani is a secreted glycoprotein modified with unique glycoforms which share a structural heterogeneity and immunological similarity with the dominant, cell surface phosphoglycolipid produced by all species of leishmania parasites. The post-translational modifications of this enzyme are structurally diverse and include standard high-mannose type N-linked glycosylations as well as a novel O-linked phosphoglycan. This dissertation encompasses the analysis of these structures including assessment of their size, hexose composition and sites of attachment to the protein. These analyses have employed both carbohydrate and protein chemistry techniques, as well as physical methods such as mass spectrometry. The N-linked glycosylations have been compared with those previously characterized on other Leishmania proteins and show substantial structural similarity. The O-linked phosphoglycans are unique to L. donovani, and are composed of phosphodisaccharides with the structure 4-O-(beta-D-galactopyranosyl)-alpha-D-mannopyranosyl-1-phosphate. These phosphodisaccharides are arranged in linear polymers by way of a phosphodiester linkage between the C1 hydroxyl of mannose and the C6 hydroxyl of galactose. Linkage of this structure to the protein is novel and proceeds via a phosphodiester to selected serine residues that are contained within a consensus protein sequence. This sequence occurs in excess of 20 times within the SAcP, which results in abundant glycan modification and contributes to the heterogeneity displayed by this enzyme. The biosynthetic machinery used to produce these structures was also investigated. The addition of phosphoglycan to the SAcP is initiated by the transfer of alpha-D-mannopyranosyl-1-phosphate from GDP-Man to the protein catalysed by a mannosyl phosphate transferase (MPT). An assay for this enzyme is described using a synthetic peptide substrate to which radiolabeled mannose can be transferred from GDP-[14C] Man. This assay has been used to partially characteriz the MPT and has assisted in the isolation of the enzyme using an affinity chromatography approach. Sequence analysis and amino acid analysis of the enzyme isolated in this way has shown that the MPT is a novel molecule that does not presently exist in the public database. / Graduate