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<p>Atherosclerosis is a complex multi-factorial disease that involves the interaction of many cell types and a plethora of molecular events. Initiation of the disease occurs when circulating LDL gets trapped in the sub-endothelial space of arteries, where LDL is oxidized causing inflammatory responses by endothelial cells. This results in recruitment and differentiation of monocytes into macrophages; macrophages in turn continue to take up cholesterol and propagate inflammation. Such a gloomy milieu of immune cells, lipids, and smooth muscle cells can give rise to atherosclerotic plaques, which then cause stenosis, vascular stiffening and eventually thrombosis. Common risk factors such as cholesterol levels, lifestyle and genetic predisposition can accelerate this potentially life threatening series of events. The downstream long-term effects of atherosclerosis, including heart disease and strokes, are now the number one cause of death in the world. While a large amount of knowledge and evidence is available in understanding this disease, prevention and treatment strategies remain somewhat ineffective. Sialylation of immune cells, lipoproteins and cellular receptors has been previously implicated in metabolic and molecular pathways relevant to atherosclerosis; however, little is known about the functional role of sialidase in these processes. Sialidase cleaves sialic acid, and is a ubiquitously expressed and evolutionarily conserved protein with essential functions in many life forms. In this study, we sought to investigate the impact of sialidase activity on atherosclerosis, emphasizing the interaction of lipid metabolism and inflammation. We have demonstrated a significant role for sialidase in cholesterol iv and lipoprotein metabolism in vivo. Specifically, hypomorphic sialidase mice have increased hepatic storage of lipids and triglycerides, decreased VLDL production, lower circulating LDL levels and alterations in regulation of LDLR. Mice over-expressing hepatic human sialidase have increased atherosclerotic lesion formation, higher serum cholesterol esters and lower levels of hepatic LDLR and SRB-1 protein. In vitro, we have shown that VLDL can induce differentiation and cytokine production in monocytes coupled with an up-regulation of Neu1. Inhibition of sialidase using DANA attenuated VLDL-induced monocyte differentiation and lipid uptake, as well as activation of macrophages, implicating Neu1 in inflammatory processes associated with initiation of atherosclerosis. Furthermore, we have shown that hypomorphic sialidase activity increases LDLR-dependent LDL uptake and cholesterol efflux to HDL in macrophages. We conclude that reduction of sialidase activity can lead to an atheroprotective phenotype with multiple effects on mechanisms involved in disease progression. This work represents novel contributions into delineating both metabolic and inflammatory processes of atherosclerosis and enables the advancement of future treatment strategies.</p> / Doctor of Philosophy (PhD)

Development of atherosclerosis, the hardening of the arteries, is dependent on levels of serum cholesterol, which is regulated by the liver via LDL receptors (LDLR). The expression and internalization of LDL receptors depend on several proteins including PCSK9. In fact, previous studies in our laboratory have shown that NEU1 down regulation leads to LDLR hypersialylation which results in its stabilization via reduced interactions with PCSK9. New evidence suggests that NEU1 which de-sialylates LDLR, may affect the ability of another hepatic receptor, the asialoglycoprotein receptor (ASGR), which is comprised of ASGR1 and ASGR2, to interact with LDLR potentially causing its internalization and therefore reduced ability to take up LDL. We investigated how sialidase plays a role in the interaction of ASGR with LDLR. Knockdown and overexpression experiments suggest that NEU1 allows stabilization of LDLR at the cell membrane via ASGR interactions. Treatment of HepG2 cells with monensin which inhibits recycling from the early endosome, unveiled a new truncated ASGR1 isoform potentially lacking its lectin motif. This may be a novel regulatory step in ASGR biosynthesis that warrants further studies. Lysosomal inhibition with chloroquine resulted in concurrent accumulations of NEU1, LDLR and ASGR1, further suggesting these proteins are biosynthetically connected. Our studies revealed a novel isoform of ASGR1 in membrane fractions of HepG2 cell lysates that can associate with NEU1 and LDLR. The impact of NEU1 and ASGR1 on the function and stability of LDLR might lead to new clues for lowering serum cholesterol and reducing atherosclerosis. / Thesis / Master of Science (MSc)

Yes / Peptidyl-prolyl isomerase (PPIase) lipoproteins have been shown to influence the virulence of a number of Gram-positive bacterial human and animal pathogens, most likely through facilitating the folding of cell envelope and secreted virulence factors. Here, we used a proteomic approach to demonstrate that the Streptococcus equi PPIase SEQ0694 alters the production of multiple secreted proteins, including at least two putative virulence factors (FNE and IdeE2). We demonstrate also that, despite some unusual sequence features, recombinant SEQ0694 and its central parvulin domain are functional PPIases. These data add to our knowledge of the mechanisms by which lipoprotein PPIases contribute to the virulence of streptococcal pathogens.

No / Metal ion acquisition and homeostasis are essential for bacterial survival, growth and physiology. A family of metal ion, ABC-type import systems have been identified in Gram-positive bacteria, in which the solute-binding proteins are predicted to be membrane-anchored lipoproteins. The prediction that the MtsA protein of Streptococcus agalactiae A909 is a lipoprotein was confirmed. The expression of MtsA was co-ordinately regulated by the presence of both manganese and ferrous ions suggesting that MtsA may be involved in the uptake of both these ions. MtsA was shown to be expressed at levels of ferrous ions known to be present in amniotic fluid, a growth medium for S. agalactiae during neonatal infection.