Sialic acids, which are the outermost sugars on the glycoproteins and glycolipids of mammalian cells, can be substituted at the 4,7,8, and 9 positions with O-acetyl esters (or other less common substitutions) in a tissue-specific fashion. However, little is known about the biological roles of thes ester groups. This proposal is a continuation of a detailed study of the biosynthesis and regulation of O-acetylated sialic acids. We have previously developed new methods to study O-acetylated sialic acids, discovered the phenomenon of O-acetyl migration, discovered neuraminidases that can release O-acetylated sialic acids and identified tissue culture cell lines that synthesize them. We have partially purified a sialic-acid-specific O-acetyl-esterase from human red blood cells and found that it is identical to the """"""""non-specific"""""""" esterased (which is tightly linked to the """"""""retinoblastoma gene"""""""" and the Wilson's disease gene on chromosome 13). We plan to further study this enzyme, particularly in retinoblastoma cells that are deficient in it, and to search for other sialic-acid-apecific esterases that could be involved in regulating O-acetylation. We have discovered a rat liver Golgi acetyl-coenzyme A transporter that provides the donor for the O-acetylation reaction; we plan to further study this transporter by affinity labelling, purification and re-constitution. We have begun studies aimed at purifying, characterizing and ultimately achieving the genetic cloning of the sialic acid-specific O-acetyltransferaseis) from E.Coli and from rat liver Golgi. We also propose approaches to generating """"""""high-acetylator"""""""" cell lines, and studies to directly demonstrate the subcellular sites of the O-acetylation reaction in such cells. The tools, probes and information generated from these studies will be used to understand the mechansims of regulation of O-acetylation in specific biological systems. For example, we have discovered that the sialic acids of rat and human colon become O-acetylated primarily after birth. We will further define the basis for this phenomenon, and pursue the hypothesis the change is induced by products arising from bacterial colonisation. In parallel studies we discovered O-acetylated GD3 (recognized by amuring monoclonal antibody D1.1) a developmentally regulated brain ganglioside also expressed in human melanoma cells. The biological basis for this expression can also be explored. A long-range goal is to use the genetic probes arising from these studies to attempt abrogation of O-acetylation in tissue-culture cells and in intact animals. These studies could have implications for the understanding of a wide variety of biological and pathological processes, such as cell surface involvement in development and malignant transformation, bacterial antigenicity and pathogenicity, alternate pathway complement activation and the generation of tumor antigens.
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