Synthesis and transport of glycoproteins and proteoglycans in the apical and basolateral secretory pathways of epithelial MDCK cells

Abstract

Sorting of newly synthesized proteins in the secretory pathway of polarized cells is governed by signals of proteinacous nature or post-translationally added structures such as glycans. Among all polarized cell types, epithelial cells are the most studied when it comes to polarized protein sorting, due to the differential access to the apical and basolateral membrane domains obtained when cells are grown on permeable supports. The most studied epithelial cell line in culture is the Madin-Darby canine kidney (MDCK) cell line, isolated from a dog kidney cortex in 1958. In the textbook view, proteins destined for the apical and basolateral membrane domains travel a common route through the secretory pathway to the trans-Golgi network, the exit site of the Golgi apparatus, from where further transport is mediated by specialized apical and basolateral transport containers. Some data obtained from studies of the soluble proteoglycan (PG) serglycin (Tveit et al. 2005; Vuong et al. 2006) and N-linked glycoproteins (Alfalah et al. 2005), however, indicate that sorting may take place early in the secretory pathway. Serglycin was modified differently in the apical and basolateral secretory routes, where basolateral passage resulted in secreted serglycin which was much more intensely sulfated than the apically secreted variant, the latter accounting for 85 % of the secreted molecules (Tveit et al. 2005). Based on these data, we wanted to study further the apical sorting capability of the serglycin molecule and the differential post-translational modification in the two pathways. Serglycin had been expressed with a green fluorescent protein (GFP) tag at the C-terminus, and for comparison, we chose to make all new variants and model proteins as GFP fusion proteins when expressed in MDCK cells. The glycosaminoglycan (GAG) attachment domain of serglycin was transferred to the junction between GFP and rat growth hormone (rGH), a non-glycosylated, non-sorted secretory protein. A variant of rGH carrying two Nglycosylation sites in addition was also expressed, since these have been shown to mediate apical sorting in MDCK cells (Scheiffele et al. 1995). The serglycin GAG domain was mainly modified with chondroitin sulfate (CS) chains and mediated apical sorting in the rGH context, but the higher sulfation intensity in the basolateral route was lost (Paper I). In fact, this capability seems to be coded by structures outside the GAG binding domain of serglycin, although not the internal disulfide bridge of the protein core (Paper III). The protein core also harbours a second type of apical sorting information of yet unidentified nature, since serglycin molecules from which all GAG attachment sites had been removed were also secreted mainly to the apical medium (paper III). We also wanted to address whether N-glycans also could be differentially processed in the apical and basolateral secretory routes of MDCK cells. To this end, we expressed rGH with two sites for N-glycosylation and a C-terminal GFP tag, and a variant with the GAG binding domain of serglycin fused between rGH and GFP in addition. The N-glycan modifications were analysed by mass spectrometry. Only marginal differences in N-glycan processing and site occupancy were observed for the model proteins after their respective apical and basolateral secretion. Insertion of the GAG attachment domain, however, influenced the synthesis of the N-glycan structures in a more acidic direction. Thus in PGs that also carry N-glycans, the GAG modification may have an impact on N-glycan structure.