The canine 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 fused to GFP was stably expressed with a typical Golgi localization in MDCK II cells (MDCK II-PAPST1). The capacity for PAPS uptake into Golgi vesicles was enhanced to almost three times that of Golgi vesicles isolated from untransfected cells. We have previously shown that chondroitin sulfate proteoglycans (CSPGs) are several times more intensely sulfated in the basolateral than the apical secretory pathway in MDCK II cells (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603). Here we demonstrate that increased availability of PAPS in the Golgi lumen enhances the sulfation of CSPG in the apical pathway several times, while sulfation of CSPGs in the basolateral pathway shows minor changes. Sulfation of heparan sulfate proteoglycans is essentially unchanged. Our data indicate that CSPG sulfation in the apical pathway of MDCK II cells occurs at suboptimal conditions, either because the sulfotransferases involved have high K(m) values, or there is a lower PAPS concentration in the lumen of the apical secretory route than in the basolateral counterpart.
SummaryA large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells. (J Histochem Cytochem 60:926-935, 2012)
PGs (proteoglycans) are proteins acquiring long, linear and sulfated GAG (glycosaminoglycan) chains during Golgi passage. In MDCK cells (Madin-Darby canine kidney cells), most of the CS (chondroitin sulfate) PGs are secreted apically, whereas most of the HS (heparan sulfate) PGs are secreted basolaterally. The apical and basolateral secretory routes differ in their GAG synthesis, since a protein core that traverses both routes acquires shorter chains, but more sulfate, in the basolateral pathway than in the apical counterpart [Tveit, Dick, Skibeli and Prydz (2005) J. Biol. Chem. 280, 29596-29603]. Golgi cisternae and the trans-Golgi network have slightly acidic lumens. We therefore investigated how neutralization of endomembrane compartments with the vacuolar H(+)-ATPase inhibitor Baf A(1) (bafilomycin A(1)) affected GAG synthesis and PG sorting in MDCK cells. Baf A(1) induced a slight reduction in basolateral secretion of macromolecules, which was compensated by an apical increase. More dramatic changes occurred to PG synthesis in the apical pathway on neutralization. The difference in apical and basolateral PG sulfation levels observed for control cells was abolished, due to enhanced sulfation of apical CS-GAGs. In addition, a large fraction of apical HS-GAGs was elongated to longer chain lengths. The differential sensitivity of the apical and basolateral secretory pathways to Baf A(1) indicates that the apical pathway is more acidic than the basolateral counterpart in untreated MDCK cells. Neutralization gave an apical GAG output that was more similar to that of the basolateral pathway, suggesting that neutralization made the luminal environments of the two pathways more similar.
Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.
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