Karen Palter
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Office: Department of Biology 343 Biological Life Sciences Building Phone: (215) 204-8854 |
Research Interests Role of sialylation during Drosophila melanogaster development The sialic acids are a family of sugars that mainly occur as terminal components of cell surface glycoproteins and glycolipids. In vertebrates, the regulated presence or absence of a polymer of sialic acid (PSA) on the neural cell adhesion molecule (NCAM) is required for the proper establishment and function of the nervous system. It has been controversial whether insects have a sialic acid pathway. Immunological and analytical reports suggest that both sialic acid and PSA are present in insects, whereas efforts to find enzymatic activity or pathway intermediates have been negative. Recently, we and others (Kole et al., 2004) identified and cloned homologs of genes encoding enzymes in the sialic acid pathway in Drosophila and showed that the sialic acid synthase, CMP-sialic synthese and sialyltransferase genes were functional in expression systems. A main project in my laboratory is to investigate whether a functional sialic pathway is present in Drosophila that results in sialylation of glycoconjugates. In addition, we are testing the hypothesis that in Drosophila, either mono or polysialylation plays an important role in the establishment of the embryonic nervous system and in the encoding of memory, similar to its role in vertebrate development. We are approaching this problem by: 1) a determination of the expression pattern of the Drosophila sialic acid synthase, CMP-sialic acid synthase and sialyltransferase throughout embryonic development and in the adult brain at both the RNA and protein levels. 2) an examination of the phenotypes of loss of function mutations of the Drosophila sialic acid synthase and siayltransferase. To eliminate gene function two approaches were used. First we selected for an imprecise excision of a P element that had inserted near the 5’ end of the sialic acid sythetase, which created a deletion of the 5’ end of the coding region. Secondly, we created transgenic flies that expressed inhibitatory RNA (RNAi) for both the sialic acid synthetase and the sialyltransferase. Flies lacking sialic acid display a range of behavioral and structural defects. These experiments should definitively establish whether the role of sialic acid in Drosophila is PSA modulation of nervous system function. If this hypothesis is shown to be correct then Drosophila may be useful as a model genetic organism to study PSA –dependent regulation of synaptic plasticity. Manipulating the glycosylation pathway in insect expression systems One project in my laboratory involves genetically engineering insect cells for the production of heterologous mammalian proteins containing the mammalian pattern of post-translational carbohydrate modifications. Although insect cells have many advantages over mammalian cells for protein expression, they currently yield proteins with very different glycosylation patterns from those generated by mammalian cells. Anomalously glycosylated proteins if used clinically would be rapidly cleared from the body in the liver and additionally may cause immunological problems. I am collaborating with Dr. Michael Betenbaugh, a biochemical engineer in the Chemical Engineering Department and Dr. Y.C. Lee and Dr. Noboru Tomiya, carbohydrate biochemists in the Biology Department at Johns Hopkins University on this project. This work is being developed for its high commercial value using both Drosophila melanogaster as a model organism, and also heterologous protein expression in lepidopteran insect cell lines using baculovirus.. I have been using the Flybase database to identify glycosylation enzymes that are shared by mammals and those that are unique to insect cells. We are examining whether insect N-glycans are shorter than those found in mammals because they possess a processing b-N-acetylhexosaminidase in the secretory pathway, which mammals lack. We postulate that this insect specific hexosaminidase removes the terminal GlcNAc from all secretory proteins that is required for the addition of the terminal galactosyl and sialic acid residues found on mammalan proteins.
Selected publications: Viswanathan, K., Tomiya, N., Park, J., Singh, S., Lee, Y.C., Palter, K.B., and Betenbaugh, M.J. (2005) Expression of a functional Drosophila CMP-sialic acid synthetase: Differential localization of the Drososphila and human enzymes. Submitted. Tomiya N, Howe D, Aumiller JJ, Pathak M, Park J, Palter KB, Jarvis DL, Betenbaugh MJ, Lee, YC. (2003). Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-N-acetylglucosaminyltransferase II. Glycobiology. 13, 23-34. Kim, K., Lawrence, S.M., Park, J., Pitts, L., Vann, W.F., Betenbaugh, M.J., and Palter, K.B. (2002). Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: Evidence for endogenous sialic acid biosynthetic ability in insects. Glycobiology 12, 73-83. |
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