PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.

Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.

Reduced sulfation of chondroitin sulfate but not heparan sulfate in kidneys of diabetic db/db mice.

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.

Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

A 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.

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment.

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.

Temporary fatigue and altered extracellular matrix in skeletal muscle during progression of heart failure in rats.

Patients with congestive heart failure (CHF) experience increased skeletal muscle fatigue. The mechanism underlying this phenomenon is unknown, but a deranged extracellular matrix (ECM) might be a contributing factor. Hence, we examined ECM components and regulators in a rat postinfarction model of CHF. At various time points during a 3.5 mo-period after induction of CHF in rats by left coronary artery ligation, blood, interstitial fluid (IF), and muscles were sampled. Isoflurane anesthesia was employed during all surgical procedures. IF was extracted by wicks inserted intermuscularly in a hind limb. We measured cytokines in plasma and IF, whereas matrix metalloproteinase (MMP) activity and collagen content, as well as the level of glycosaminoglycans and hyaluronan were determined in hind limb muscle. In vivo fatigue protocols of the soleus muscle were performed at 42 and 112 days after induction of heart failure. We found that the MMP activity and collagen content in the skeletal muscles increased significantly at 42 days after induction of CHF, and these changes were time related to increased skeletal muscle fatigability. These parameters returned to sham levels at 112 days. VEGF in IF was significantly lower in CHF compared with sham-operated rats at 3 and 10 days, but no difference was observed at 112 days. We conclude that temporary alterations in the ECM, possibly triggered by VEGF, are related to a transient development of skeletal muscle fatigue in CHF.

Neutralization of endomembrane compartments in epithelial MDCK cells affects proteoglycan synthesis in the apical secretory pathway.

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.

Overexpression of the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells.

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.