Identification of Functional Domains in Sphingomyelin Synthase (SMS) and Further Investigation into the Role of Phospholipid Transfer Protein (PLTP) in Lipid Metabolism and Atherosclerosis

Abstract

Lipid metabolism is closely related to the development of atherosclerosis. The work presented in this thesis involves two genes which are important for lipid metabolism and atherosclerosis: sphingomyelin synthase (SMS) and phospholipid transfer protein (PLTP). Sphingomyelin synthase (SMS) utilizes ceramide and phosphatidylcholine as substrates to produce sphingomyelin (SM) and diacylglycerol, thereby regulating important lipid messengers. There are two isoforms of the enzyme, SMS1 and SMS2. It has been speculated that the active site of both SMS1 and SMS2, containing two evolutionary conserved histidines and one aspartic acid, is identical to that of lipid phosphate phosphatase (LPP). We systematically mutated these amino acids by site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. Despite sharing 77% sequence homology to each other, SMS1 is located in the trans-Golgi while SMS2 is located in the plasma membrane as well as the Golgi. This prompted us to explore what targeting signals exist in SMS1 and SMS2. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. Therefore, we determined the targeting signals in SMS1 and SMS2 through a combination of creating truncations of non-homologous amino acids from SMS1 and SMS2 and creating SMS chimeras by swapping non-homologous amino acid sequences between SMS1 and SMS2. We found that SMS1 and SMS2 transit through the classical secretory pathway. Furthermore, SMS1 contains a C-terminal Golgi targeting signal and SMS2 contains a C-terminal plasma membrane targeting domain. (PLTP) is an ubiquitously expressed, secreted protein which functions in the blood and in tissues. A high saturated fat diet induces free cholesterol and phospholipid accumulation in the plasma of phospholipid transfer protein (Pltp) deficient mice. We examined the atherogenic consequence of this phenomenon and investigated the possible mechanism(s). To study atherosclerosis, we bred Pltp KO mice with Apoe KO mice, a mouse model of atherosclerosis. Pltp KO/Apoe KO mice, fed a coconut oil-enriched high-fat diet (COD) for 7 weeks, had higher plasma free cholesterol (149%), phospholipids (15%), and sphingomyelin (54%) than Apoe KO controls. In contrast to chow-fed animals, COD-fed Pltp KO/Apoe KO mice had the same atherosclerotic lesion size as that in Apoe KO mice. Similar to Pltp KO mice, plasma from COD fed Pltp KO/Apoe KO mice contained VLDL/LDL sized lamellar particles. Bile measurement indicated that COD-fed PltpKO mice have 33% less hepatic cholesterol output than controls. In conclusion, COD-fed Pltp deficient mice are no longer protected from atherosclerosis and have impaired biliary lipid secretion associated with free cholesterol and phospholipid accumulation.