Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and Sorting in Filter-Grown and Polarized...

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with ³⁵S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.