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.