Synthesis and transport of Syndecan 1 and 4 variants in MDCK cells

Syndecans (Syns) are transmembrane heparan sulfate proteoglycans (HSPGs) and comprise a family of four members, Syn 1-4. Syns have important roles as cell surface receptors involved in cell-cell and/or cell-matrix interactions. Syn 4 is expressed in nearly all cell types and tissues, and is involved in focal adhesion. In this thesis, DNA coding green fluorescent protein (GFP) was fused into Syn 4 in the extracellular domain after amino acid (aa) 84, to allow observation of the expressed recombinant protein in the confocal microscope. GFP has a molecular mass of 26.9 kDa and could potentially interfere with sorting of Syn 4. Therefore, a smaller tag was also used as an alternative; a Hemagglutinin (HA; 9aa) tag was inserted after aa 84 instead of GFP. Syn 4-GFP or Syn 4-HA were then expressed in Madin-Darby canine kidney (MDCK) II cells by stable transfection, followed by examination of plasma membrane localization, proteolytic shedding, the nature of the GAGs attached to Syn 4 protein cores, and the formation of Syn dimers and monomers. We also took advantage of a previously generated MDCK II cell line expressing Syn 1-GFP, to compare the synthesis and transport of this recombinant proteoglycan (PG) with the Syn 4 variants. Membrane biotinylation studies of filter grown MDCK II cells expressing Syn 4-GFP or Syn 4-HA and studies of shedding with radioactive [35S]-Cysteine/methionine (Cys/Met) labeling and Western blotting revealed variability in the results between these two constructs. Syn 4-GFP was mostly shed into the Apical (Api) medium, while shed Syn 4-HA was mostly found in the Baso medium. The type of GAG chains attached to Syn 4 core proteins was examined by enzymatic degradation of heparan sulfate (HS) GAG chains with heparitinases (Hep) and chondroitin sulfate (CS) GAG chains with chondroitinase ABC (cABC) after metabolic labeling of transfected MDCK II cells with [35S]-sulfate or [35S]-Cys/Met and by Western blotting. The Hep treatment gave a dramatic decrease in the amount of [35S]-sulfate in the immunoisolated PGs, indicating that HS chains were the GAGs predominantly attached to the isolated Syn. Syn 4-GFP appears only in a monomeric form in SDS-PAGE analysis, while Syn 4-HA appears mainly as a dimer. This finding might have important consequences for the differences observed in cellular localization of the two Syn 4 variants

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes

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Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes

<p>The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an <i>in vitro</i> normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.</p