The 2,8-linked polysialic acid (polySia) glycotope is involved in cell migration and cell adhesion and thus plays a central role in the regulation of mammalian developmental processes. It is also an important tumor-associated oncodevelopmental antigen on a number of human cancers. On neurotropic E. coli K1 and Neisseria meningitidis Gp.B, the polySia capsule is a neurovirulent determinant associated with neonatal meningitis in humans. Therefore, it is important to understand the molecular mechanism of polySia chain synthesis, and how synthesis is controlled. These are the long-term objectives of our studies. The enzymes responsible for synthesis of polySia ar the CMP-Sia: 2,8 polysialytransferase (polySTs), designated PST and STX in mammals and NeuS in E. coli K1. There is no primary sequence homology between the mammalian and bacterial polySTs, yet all three catalyze synthesis of identical polySia chains. A key hypothesis to be tested is that PST, STX and NeuS achieve the same catalytic functions by rearranging conserved amino acid motifs spatially in their active sites to bind CMP-Sia and initiate polySia chain synthesis while bound to the enzyme.
The Specific Aims of this grant are three-fold.
Aim 1 will characterize by biochemical methods the length of the polySia chains synthesized by membrane-bond and soluble constructs of PST and STX derived from transfected N-CAM(+) and (-) cells. The nature of the potential reducing terminus in N-CAM (-)/polySia(+) transfected cells will als be determined.
Aim 2 includes structure-function studies that will investigate the structural basis for polysialylation. Active site cysteine and conserved basic amino acid motifs implicated in catalysis and processivity will be studied by site-directed mutagenesis.
Aim 3 will use in vitro reconstitution experiments to determine, """"""""how many enzymes does it take to synthesize polySia?"""""""". Successful completion of these studies will provide basic information for understanding the molecular mechanism of polysialylation, and the critical domains of the polySTs involved. This will also have important implications for basic studies relating to neural development and plasticity, neoplastic disease and molecular microbiology and pathogenesis, and provide a necessary framework for future studies.
