OpenUCT :: Browsing by Department "Division of Medical Biochemistry"

Browsing by Department "Division of Medical Biochemistry"

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Open Access
The 341C/T polymorphism in the GSTP1 gene is associated with increased risk of oesophageal cancer
(BioMed Central Ltd, 2010) Li, Dongping; Dandara, Collet; Parker, M Iqbal
BACKGROUND: The Glutathione S-transferases (GSTs) comprise a group of enzymes that are critical in the detoxification of carcinogens. In this study the effects of polymorphisms in these genes on the risk of developing oesophageal squamous cell carcinoma (OSCC) were evaluated in a hospital-based case-control study in two South African population groups. Genetic polymorphisms in GSTs were investigated in 245 patients and 288 controls samples by PCR-RFLP analysis. RESULTS: The GSTP1 341T variant was associated with significantly increased risk of developing OSCC as observed from the odds ratios for the GSTP1 341C/T and GSTP1 341T/T genotypes (OR = 4.98; 95%CI 3.05-8.11 and OR = 10.9; 95%CI 2.43-49.1, respectively) when compared to the homozygous GSTP1 341C/C genotype. The risk for OSCC in the combined GSTP1 341C/T and T/T genotypes was higher in tobacco smokers (OR = 7.51, 95% CI 3.82-14.7), alcohol consumers (OR = 15.3, 95% CI 1.81-12.9) and those using wood or charcoal for cooking and heating (OR = 12.1, 95% CI 3.26-49) when compared to those who did not smoke tobacco, or did not consume alcohol or user other forms of fuel for cooking and heating. Despite the close proximity of the two GSTP1 SNPs (313A>G and 341C>T), they were not in linkage disequilibrium in these two population groups (D':1.0, LOD: 0.52, r2: 0.225). The GSTP1 313A/G polymorphism on the other hand, did not display any association with OSSC. The homozygous GSTT1*0 genotype was associated with increased risk of OSCC (OR = 1.71, 95%CI 1.18-2.46) while the homozygous GSTM1*0 genotype was associated with significantly decreased risk of OSCC in the Mixed Ancestry subjects (OR= 0.39, 95%CI 0.25-0.62). CONCLUSIONS: This study shows that the risk of developing OSCC in the South African population can be partly explained by genetic polymorphisms in GST coding genes and their interaction with environmental factors such as tobacco smoke and alcohol consumption.
Open Access
A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase
(2001) Alfalah, Marwan; Parkin, Edward T; Jacob, Ralf; Sturrock, Edward D; Mentele, Reinhard; Turner, Anthony J; HOOPER, Nigel M; Naim, Hassan Y
Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
Open Access
Altered protein expression patterns in oesophageal cancer
(2009) Zemanay, Widaad; Hendricks, Denver
Oesophageal squamous cell carcinoma presents a significant health burden in South Africa. It is one of the most common causes of cancer-related mortality of South African black males, as a result of its asymptomatic progression leading to late diagnosis and poor prognosis. The aim of this study was to identify membrane or membrane-associated proteins that are expressed at different levels in oesophageal tumour tissue when compared to normal tissue. The identification of such proteins would be an important step towards the development of better diagnostic and therapeutic strategies for this disease. Two proteomic approaches, were employed to identify differentially expressed proteins.
Open Access
An angiotensin I-converting enzyme mutation (Y465D) causes a dramatic increase in blood ACE via accelerated ACE shedding
(Public Library of Science, 2011) Danilov, Sergei M; Gordon, Kerry; Nesterovitch, Andrew B; Lünsdorf, Heinrich; Chen, Zhenlong; Castellon, Maricela; Popova, Isolda A; Kalinin, Sergey; Mendonca, Emma; Petukhov, Pavel A
BACKGROUND: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. METHODOLOGY/PRINCIPAL FINDINGS: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).
Open Access
Angiotensin-converting enzyme cleavage of the Alzheimer's beta-amyloid peptide
(2015) Larmuth, Kate Morgan; Sturrock, Edward D
Angiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (N and C) with different substrate specificities. ACE is a central component of the intrinsic brain renin angiotensin-aldosterone system (BRAAS), well renowned as the regulator of blood pressure. The BRAAS has alternate functions that extend beyond fluid and blood pressure homeostasis into areas such as neurological function. As a result, it is implicated in many neurodegenerative diseases including Alzheimer's disease (AD). ACE's specific mechanistic role in AD is not entirely clear and is somewhat controversial. However, it has been shown that ACE hydrolyses the amyloid beta (Aβ) peptide, the putative causative agent of AD. This study aimed to investigate the molecular basis of ACE hydrolysis of Aβ by determining : 1) the kinetic parameters of five different forms of human ACE with various N-terminal amyloid beta (Aβ) substrates; 2) the specific active site determinants of Aβ-domain selectivity; and 3) the high-resolution crystal structures of the N-domain of ACE in complex with Aβ(1-16), Aβ(10-16), Aβ(4-10), the FRET Aβ(4-10)Y and Aβ(35-42) peptides. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, Aβ(1-16 ) was preferentially cleaved by the truncated N-domain; however, the presence of an inactive C-domain in full-length ACE greatly reduced enzyme activity and affected domain-selectivity. Two fluorogenic substrates, designed specifically to assess ACE's mechanism of Aβ hydrolysis Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7/Ser8 bond. The Aβ(4-10)Q peptide was a poor substrate of ACE but was N-selective, with a selectivity driven largely by interactions with the domain-specific residues of the S2 and S2' pockets. The selectivity of the S2' residues were confirmed with a similar, more physiological, fluorogenic Aβ(4-10)Y peptide. This work provides further understanding towards the substrate determinants of N-selectivity, highlighting the importance of the S2' Ser357. ACE C-domain hydrolysed Aβ(4-10)Y with modest efficiency compared to the other substrates, where hydrolysis under the same conditions did not occur. Moreover, Aβ(4-10)Y also displayed N-domain selectivity. In contrast to Aβ(1-16) and Aβ(410)Q, both sACE and the double C-domain (CC-sACE) construct showed positive domain cooperativity towards Aβ(4-10)Y. The high-resolution crystal structures of the N-domain in complex with five Aβ peptide fragments provided an overlapping, conserved, molecular mechanism of peptide binding and evidence of the enzyme's broad exoprotease activity. In addition to the kinetic and structural studies, ACE's signalling response to the N-selective Aβ(1-16) and Aβ(1-42) was investigated using immunodetection and mass spectrometry. Similar to the ACE inhibitor lisinopril, the Aβ peptides elicited ACE signalling by phosphorylation of the cytoplasmic Ser1270 residue and JNK activation. The signalling response of ACE was coupled to increased ACE activity an d expression on treatment with Aβ(1-42). These studies allowed us to rationalise the increased ACE activity and expression found in AD, may arise through direct interactions with Aβ. This work provides a kinetic, structural and mechanistic understanding of the selective cleavage of Aβ by the N and C catalytic sites of ACE. Due to the broad substrate specificity of the two domains of ACE, and the overarching N- selectivity of Aβ hydrolysis, these findings provide rationale for further in vivo pharmacological studies on the mechanism of action C- domain-selective inhibitors, in the context of AD.
Open Access
Anti-oesophageal cancer activity in extracts of deep-water Marion Island sponges
(2005) Davies-Coleman, Michael T; Froneman, William; Keyzers, Robert; Whibley, Catherine; Hendricks, Denver; Samaai, Toufiek; McQuaid, Christopher
OESOPHAGEAL CANCER IS ONE OF THE most common causes of cancer-related deaths in South African black males. The limited efficacy of chemotherapeutic agents to treat this disease has prompted a search for potential new chemical entities with anticancer properties. We report here on the evidence for anti-oesophageal cancer activity in the methanolic extracts of five species of sponges dredged from a depth of approximately 100 m in the vicinity of Marion Island in the Southern Ocean during the autumn of 2004.
Open Access
Antigenic mimicry and autoantibodies in rheumatic fever
(1990) Eichbaum, Quentin Gavin
Open Access
Applications of a radioimmunoassay technique to the study of luteinizing hormone secretion in the rat
(1975) Querido, David; Beardwoor, C J
A sensitive and reproducible double antibody radioimmunoassay technique, requiring 50ul of unknown serum or plasma per assay tube, is described for use with 125I and rabbit anti-rat LH serum. The assay system was applied to the study of LH secretion in rats under both normal and experimentally manipulated conditions. Particular attention was focussed upon comparison of circulating LH levels in conscious, unstressed animals with those in anaesthetized animals, with or without surgical stress. Thereafter, the effects of acoustic stimulation and of exogenous LRH administration were studied in conscious and anaesthetized animals. Urethane anaesthesia exerted a profound effect upon the LR-secretory response to exogenous LRH in male rats. Available evidence suggests that the blood sampling method, surgical stress and anaesthesia are each capable of significantly influencing LH secretion, thereby emphasizing the value of studies using conscious, unstressed animals. While a direct effect of urethane on the pituitary gland cannot be excluded, attention is drawn to the possible mediation of a urethane-sensitive inhibitory influence in the mechanism controlling LH secretion in the rat.
Open Access
Bacterial disease and antimicrobial susceptibility patterns in HIV-infected, hospitalized children: a retrospective cohort study
(Public Library of Science, 2008) Jaspan, Heather B; Huang, Lyen C; Cotton, Mark F; Whitelaw, Andrew; Myer, Landon
BACKGROUND: Serious bacterial infections are a major source of morbidity and mortality in HIV-infected children. The spectrum of disease is wide, and responsible organisms vary according to setting. The use of antibiotic prophylaxis and the emergence of multi-drug resistant bacteria necessitate examination of responsible organisms and their antibiotic susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective cohort study of all HIV-positive pediatric admissions at an urban public sector hospital in Cape Town between January 2002 and June 2006 was conducted. Children between the ages of one month and nine years with laboratory confirmed HIV status, serious bacterial infection, and a hospital length of stay of 5 days or more, were eligible for inclusion. Organisms isolated from blood, urine, and cerebral spinal fluid cultures and their antimicrobial susceptibility were examined, and compared according to timing of isolation to distinguish nosocomial versus community-acquired. One hundred and forty-one children were identified (median age 1.2 years), 39% of whom were on antiretrovirals started before or during this hospitalization. Bacterial infections involved all organ systems, however pneumonia was most common (67%). S. pneumoniae and S. aureus were the most common gram positive and K. pneumoniae was the most common gram negative organism. K pneumoniae isolates were resistant to many first and second line antibiotics, and were all considered nosocomial. All S. aureus isolates were methicillin resistant, some of which were community-acquired. Conclusions/Significance Bacterial infections are an important source of co-morbidity in HIV-infected children in resource-limited settings. Clinicians should have a low threshold to initiate antibiotics in children requiring hospitalization. Broad-spectrum antibiotics should be used judiciously. Clinicians caring for HIV-infected children should be cognizant of the most common organisms affecting such children, and of their local antimicrobial susceptibilities, when treating empirically for serious bacterial infections.
Open Access
Baseline Predictors of Sputum Culture Conversion in Pulmonary Tuberculosis: Importance of Cavities, Smoking, Time to Detection and W-Beijing Genotype
(Public Library of Science, 2012) Visser, Marianne E; Stead, Michael C; Walzl, Gerhard; Warren, Rob; Schomaker, Michael; Grewal, Harleen M S; Swart, Elizabeth C; Maartens, Gary
BACKGROUND: Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes. Aim To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis. Design Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable. RESULTS: 113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype. CONCLUSION: We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.
Open Access
Beta-lactam antibiotic resistance in enterobacter cloacae isolated from Groot Schuur Hospital inpatients
(1991) Saunders, G L (Geoffrey Lance)
Open Access
The biochemical and molecular characterisation of respiratory mucins in TB
(2006) Govender, Ureshnie; Mall, Anwar
The role of the dominant respiratory mucins (MUC5AC and MUC5B) and MUC2 has been investigated in chronic airway diseases as it is the mucin glycoprotein that confers upon mucus its biological, rheological and physicochemical properties. Within South Africa, specifically the Western Cape, TB has wreaked havoc especially amongst those of the lower socioeconomic groups. However, despite the prevalence of the disease in South Africa and the known morbidity and mortality associated with mucus and mucin hypersecretion in respiratory diseases, little is known of the association between respiratory mucins and TB. This is a novel study that investigated the association between respiratory mucins and TB at a biochemical and molecular level.
Open Access
The biochemical characterisation of respiratory mucus & mucins in normal, asthma & COPD
(2009) Trimmel, Astrid Joan; Mall, Anwar
Airway mucus hyper-secretion is the main cause of mortality and morbidity in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) and is the leading cause of death in South Africa. Mucus is a viscid, slimy visco-elastic gel-like material, which coats the epithelial tissue of gastrointestinal, reproductive and respiratory tracts. Mucus has defined rheological properties that enable it to be transported out of the lungs by mucociliary clearance.
Open Access
Biogenesis of lysosomes in macrophages : intracellular pathway of lysosomal membrane protein to lysosomes
(2008) Ebrahim, Roshan; Thilo, L
Includes abstract. Includes bibliographical references (leaves 212-248).
Open Access
Characterisation of CIS- and trans-acting factors that regulate the human alpha 2(1) procollagen gene
(1999) Masemola, Agatha Maripanyane; Parker, Iqbal
The differential expression of the a2(I) procollagen gene in normal and transformed human fibroblasts has been correlated with differential in vitro DNA-protein interactions on the basal promoter region between -100 and -67. A 23 bp region of the a.2(1) procollagen promoter encompassing the G/CBE (CCTCCATTGG) and the Ctv'IE (GGAGGCCCTTTT) has previously been shown to engage in specific DNA protein interactions that determined the transcriptional activity of the promoter. The CME forms two distinct DNA-protein complexes that might be crucial in the regulation of the a2(I) procollagen gene in a cell specific manner. The hypothesis was, therefore, that depending on the protein that participates in complex formation with the CME, the gene would be activated or repressed. The objective of this study was to investigate the role of this 23 bp region in the regulation of expression. of the a2(I) procollagen gene in transformed fibroblasts. In addition, the study sought to establish the role of the proto-oncogene c-fos-in the regulation of expression of the a2(I) procollagen gene. In contrast to previous observations, this study demonstrated that only one DNA protein complex is formed on the CME and the second complex is a specific proteolytic cleavage of the product of the larger complex. Preparation of nuclear extracts in the absence of protease inhibitors, specifically leupeptin, resulted in the formation of a smaller complex, previously shown to bind the CME. The importance of this proteolytic fragment that still retains DNA binding activity is yet to be determined. In addition, the CME binding proteins were fairly ubiquitously expressed in both a.2(1) collagen producing and non-producing cells. CT-1 fibroblasts (transformed by y-irradiation) synthesise over 80% of total a2(I) collagen produced by its untransformed counterpart (WI-38 fibroblasts), whereas the gene, is down regulated in the human embryonic lung fibroblasts transformed with SV40 (SVWI-38 fibroblasts). These cell lines are therefore ideal for studying regulation of a.2(I) procollagen gene. To analyse the importance of the G/CBE and CME regions of the a.2(1) procollagen gene promoter, point mutations were introduced by site-directed mutagenesis. Mutated promoter DNA was cloned into a p8CAT reporter vector, and the activity of the promoter determined in transient transfection experiments. Mutations introduced in the G/CBE region of the a.2(1) procollagen promoter resulted in a 3-12-fold decrease in the activity of the promoter. The decrease was observed with both proximal (-343 bp) and basal (-107 bp) promoter constructs~ a significant reduction in promoter activity was observed in both CT-1 and SVWI-38 fibroblasts. These results imply that the G/CBE region of the promoter is required for the activation of transcription of the a.2(1) procollagen gene and therefore the factor that interacts with the G/CBE functions as a transcriptional activator. Previously, this factor was shown to complex with antibodies raised against the mouse CCAAT binding factor (CBF), suggesting that the protein belongs to the CBF family of transcription factors. Furthermore, these results demonstrate that the adjacent, upstream inverted GGAGG sequence is crucial for activation of the gene through the CCAAT binding element. The inhibition of promoter activity in constructs with a mutated G/CBE element was correlated with lack of protein binding to the mutated sequence as confirmed by electrophoretic mobility shift assays. Transfection of a.2(1) procollagen promoter constructs containing mutations in the CME region, however, resulted in a significant increase in promoter activity in both CT-1 and SVWI-38 fibroblasts. A much higher increase, 3-fold, was observed for the SVWI-38 cell line compared to a 1.5-fold increase observed for CT-1 fibroblasts. These results suggested that the factor that interacts with the CME functions as a repressor of the a.2(1) procollagen gene. Interestingly, the promoter activity in SVWI38 fibroblasts transfected with mutated CME constructs was similar to that observed in CT-1 fibroblasts transfected with the wild type promoter construct. An interesting observation was that repression of the a.2(1) procollagen gene via the CME required upstream elements since transfection of the basal mutated promoter did not result in increased promoter activity. From these results, it can be concluded that the CME binding protein is involved in cell-specific repression of the a2(I) procollagen gene and that the mechanism of repression appears to be dependent on the presence of upstream elements. Mutations in the G/CBE and CME pointed out the significance of these elements in the expression of the a2(I) procollagen gene and since a number of studies have characterised the mouse CCAAT binding protein, this study focused on purification and identification of the CME binding protein(s). Purification was performed by conventional biochemical techniques using heparin-agarose and sequence-specific DNA affinity chromatography, as well as separation on SDS-polyacrylamide gels. Two cycles of DNA affinity chromatography yielded two polypeptides with apparent molecular weights of 50 and 67 kDa. Automated N-terminal sequencing of the polypeptides indicated that they were blocked and therefore no sequence could be obtained. In addition, these polypeptides failed to raise an immune response in mice and rabbits. Subsequently, polypeptides were digested with trypsin in situ in polyacrylamide gels and the eluted peptides were analysed by MAWITOF-mass spectrometry. The mass:charge ratios (mlz ratios) obtained were used to search the database using a mass tolerance of 1.5 Da and only one hit was obtained. The match obtained was that of a mouse zinc finger protein of which not much is known, except that it might be a transcription factor. This result supports previous observations of Collins et al (J Cell Biochem 1998, 70: 455-467) that complex formation requires the presence of zinc. The primary structure of the CME binding protein remains to be determined. Transformation of fibroblasts is normally accompanied by changes in the expression of extracellular proteins, including type I procoUagen. Although CT-I fibroblasts, show very little change in a2(I) procollagen gene expression, the c-f os gene is drastically down-regulated. This study sought to establish if there is any relationship between the unusually high levels of the a2(I) procollagen gene in this transformed cell line and failure of the cells to stimulate c-fos expression in response to serum. CT-1 :fibroblasts that overexpressed wild type Fos were established and changes in the expression of the a,2(1) procollagen gene were measured. Overexpression of Fos down-regulated the a,2(1) procollagen gene, which was not due to increased turnover of the a1(I) procollagen mRNA. Analysis of promoter activity showed that the promoter and first intron, which has been reported to contain negative regulatory elements, did not harbour any Fas-responsive elements. The -343 bp and the -2300 bp promoter constructs were transactivated in cells overexpressing Fos. Thus, although overexpression of Fos resulted in a significant decrease in the levels of the a2(1) procollagen WA, it does not involve the region between -2300 bp and +1800 bp of the a2(1) procollagen gene. Furthermore, there was no change in the stability of the message, indicating that constitutive expression of Fos did not activate a factor that could play a role in altered turnover of the a2(I) procollagen mRNA. It is therefore possible that constitutively high levels of Fos may trigger the expression of a number of other genes, which have a negative impact on the expression of the a2(I) procollagen gene.
Open Access
Characterisation of the ectodomain shedding of angiotensin-converting enzyme
(2003) Woodman, Zenda; Sturrock, Edward D
Bibliography: leaves 240-266.
Open Access
The characterisation of the ectodomain shedding of the low density lipoprotein receptor
(2009) Parker, Ayesha; Sturrock, ED
Includes abstract. Includes bibliographical references (leaves 142-155).
Open Access
The characterisation of the peanut agglutinin an evolved plant lectin, with improved specificity to the Thompson Freidenriech antigen
(2013) Lagardien, Zaida; Blackburn, Jonathan
Peanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA).
Open Access
Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme
(2014) Conrad, Nailah; Sturrock, Edward D; Schwager, Sylva L U
Angiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE.
Open Access
Characterization of avipoxviruses for use in recombinant vaccines
(1992) Kow, Daria Karen; Dumbell, K R
Pox viruses have been demonstrated in over 60 types of wild and exotic birds as well as domestic birds. Avipox viruses have been isolated and characterised from fowls, quails, canaries, parrots and lovebirds. This work describes the first isolation of a poxvirus from Jackass penguins (Spheniscus dermersus) and the characterisation of the virus as a separate species of penguinpox virus.