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K Prydz

Publications and source records attributed to K Prydz.

At least 19 recordsLinked to original sources
PubMed2001

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.

Animals↗
PubMed2001

Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells.

Reduction of the cholesterol level in membranes of epithelial Madin-Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357-1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.

Animals↗
PubMed2001

The AGE product N epsilon-(carboxymethyl)lysine serum albumin is a modulator of proteoglycan expression in polarized cultured kidney epithelial cells.

AIMS/HYPOTHESIS: Changes in kidney function in diabetes could be due to changes in the kidney basement membranes. Proteoglycans are important constituents of this kidney extracellular matrix. This study explored the possibility that advanced glycation end products affect proteoglycan synthesis in cultured kidney epithelial cells. METHODS: Madin Darby Canine Kidney (MDCK) epithelial cells were cultured with either low glucose (5 mmol/l), low glucose with 10 micrograms/ml of N epsilon-(carboxymethyl)lysine bovine serum albumin (CML-BSA) or high glucose (25 mmol/l). From day 7-8 cells were labelled with either [35S]sulphate or [3H]glucosamine for 24 h. Labelled macromolecules were purified by gel and ion exchange chromatography, and isolated proteoglycans analysed by gel chromatography and electrophoresis. RESULTS: The CML-BSA treatment reduced the proteoglycan synthesis in MDCK cells. Neither the type of glycosaminoglycan chains made nor the molecular size of the chains was affected. CONCLUSION/INTERPRETATION: At concentrations found in the plasma of diabetes patients CML-BSA, decreases proteoglycan expression in kidney epithelial cells. Advanced glycation end products could, accordingly, promote pathological changes in kidneys of diabetics.

Animals↗
PubMed2001

Proteoglycan synthesis is increased in cells with impaired clathrin-dependent endocytosis.

Overexpression of a GTPase deficient dynamin mutant in HeLa dynK44A cells causes a block in clathrin-dependent endocytosis. When endocytosis is inhibited, these cells incorporate higher levels of [(35)S]sulfate into both cellular and secreted macromolecules and larger amounts of proteoglycans such as syndecan and perlecan are immunoprecipitated from [(35)S]sulfate-labelled lysates. Gel filtration and ion-exchange chromatography revealed that the increased [(35)S]sulfate incorporation into proteoglycans was not due to significant differences in size or density of negative charge of glycosaminoglycan chains attached to proteoglycan core proteins. On the other hand, measurements of the syndecan-1 mRNA level and of [(3)H]leucine-labelled perlecan after immunoprecipitation supported the idea that the increased [(35)S]sulfate incorporation into proteoglycans was due to a selective increase in the synthesis of proteoglycan core proteins. Interestingly, the activity of protein kinase C was increased in cells expressing mutant dynamin and inhibition of protein kinase C with BIM reduced the differences in [(35)S]sulfate incorporation between cells with normal and impaired clathrin-dependent endocytosis. Thus, the activation of protein kinase C observed upon inhibition of clathrin-dependent endocytosis may be responsible for the increased synthesis of proteoglycans.

Animals↗
PubMed2000

Lithocholic acid and sulphated lithocholic acid differ in the ability to promote matrix metalloproteinase secretion in the human colon cancer cell line CaCo-2.

The human colon carcinoma cell line CaCo-2 has the ability to sulphate the secondary bile acid lithocholic acid (LA), whereas other primary or secondary bile acids were not sulphated [Halvorsen, Kase, Prydz, Gharagozlian, Andresen and Kolset (1999) Biochem. J. 343, 533--539]. To study the biological implications of this modification, CaCo-2 cells were incubated with either LA or sulphated lithocholic acid (3-sulpholithocholic acid, SLA), and in some experiments with taurine-conjugated lithocholic acid. Increased secretion of matrix metalloproteinases (MMPs) correlates with transformation of colon epithelial cells. When CaCo-2 cells were incubated with LA, the secretion of MMP-2 was found to increase approx. 60% when analysed by gelatin zymography, and 80% when analysed by Western blotting. SLA, in contrast, did not affect the level of MMP-2 secretion, and after zymography the level of enzyme activity was 78% of control values after 18 h incubation. The secretion of MMPs is linked to increased cellular invasion and, in tumours, to increased capacity for metastasis. The ability of CaCo-2 cells to invade in a chamber assay was stimulated after exposure to LA, whereas SLA-treated cells did not differ from control cells. LA therefore seems to induce a more invasive CaCo-2 cell phenotype, as judged by the two parameters tested, whereas the sulphated counterpart, SLA, did not have these effects. Sulphation of LA in the colon may be an important mechanism to decrease the potential LA has to promote a malignant epithelial phenotype.

Bile Acids and Salts↗
PubMed2000

Synthesis and sorting of proteoglycans.

Proteoglycans are widely expressed in animal cells. Interactions between negatively charged glycosaminoglycan chains and molecules such as growth factors are essential for differentiation of cells during development and maintenance of tissue organisation. We propose that glycosaminoglycan chains play a role in targeting of proteoglycans to their proper cellular or extracellular location. The variability seen in glycosaminoglycan chain structure from cell type to cell type, which is acquired by use of particular Ser-Gly sites in the protein core, might therefore be important for post-synthesis sorting. This links regulation of glycosaminoglycan synthesis to the post-Golgi fate of proteoglycans.

Amino Acid Sequence↗
PubMed1999

Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate.

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate. Metabolically [(3)H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by approximately 70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3'-phosphoadenosine 5'-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.

Animals↗
PubMed1999

Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2.

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [(35)S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [(35)S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [(35)S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of (35)S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 microg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.

Bile Acids and Salts↗
PubMed1999

Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.

Animals↗
PubMed1999

Characterization of fibroblast growth factor 1 binding heparan sulfate domain.

Fibroblast growth factors FGF-1 and FGF-2 mediate their biological effects via heparan sulfate-dependent interactions with cell surface FGF receptors. While the specific heparan sulfate domain binding to FGF-2 has been elucidated in some detail, limited information has been available concerning heparan sulfate structures involved in the recognition of FGF-1. In the current study we present evidence that the minimal FGF-1 binding heparan sulfate sequence comprises 5-7 monosaccharide units and contains a critical trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. N-Sulfated heparan sulfate decasaccharides depleted of FGF-1 binding domains showed dose-dependent and saturable binding to FGF-2. These data indicate that the FGF-1 binding domain is distinct from the minimal FGF-2 binding site, previously shown to contain an IdoA(2-OSO3) residue but no 6-O-sulfate groups. We further show that the FGF-1 binding heparan sulfate domain is expressed in human aorta heparan sulfate in an age-related manner in contrast to the constitutively expressed FGF-2 binding domain. Reduction of heparan sulfate O-sulfation by chlorate treatment of cells selectively impedes binding to FGF-1. The present data implicate the 6-O-sulfation of IdoA(2-OSO3)-GlcNSO3 units in cellular heparan sulfate in the control of the biological activity of FGF-1.

Animals↗
PubMed1999

Apical secretion of chondroitin sulphate in polarized Madin-Darby canine kidney (MDCK) cells.

Sugar moieties have been shown to contain sufficient and necessary information to target examples of secreted and transmembrane glycoproteins to the apical surface of epithelial MDCK cells. We have investigated if the sugar chains of proteoglycans, the glycosaminoglycans, also contain structural determinants for apical transport. Here we show that although 75% of the proteoglycan secretion from MDCK cell monolayers is into the basolateral medium, 75% of the proteoglycans of the chondroitin sulphate type are secreted apically. The sorting information in the chondroitin sulphate proteoglycans is localized to the sugar chains, since protein-free chondroitin sulphate chains, initiated on hexyl beta-D-thioxyloside, were also predominantly secreted to the apical medium.

Animals↗
PubMed1998

Endocytosed ricin and asialoorosomucoid follow different intracellular pathways in hepatocytes.

Earlier studies have suggested that fluid phase endocytosis in rat hepatocytes takes place via a clathrin-independent mechanism [1,2]. This observation suggests that a relatively large amount of plasma membrane outside coated pits may be involved in hepatic endocytosis. Ricin, which binds to galactose residues on glycoproteins and glycolipids, has, in this report, been used as a general marker for the plasma membrane of hepatocytes. The endocytosis of ricin was compared with that of asialoorosomucoid (AOM) which is taken up exclusively via clathrin-coated pits. Hypertonic medium has been shown to inhibit uptake via coated pits more effectively than clathrin-independent uptake [3-5]. It was found, in this study, that the addition of 100 mM sucrose to the incubation medium inhibited the uptake of 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) more extensively than that of 125I-tyramine-cellobiose-ricin (125I-TC-ricin), compatible with the notion that the two probes are internalised via different mechanisms. Subcellular fractionation experiments indicated that 125I-TC-ricin entered a denser endocytic organelle than that receiving 125I-TC-AOM. To determine whether the separation of the two probes was due to a different transport kinetics (i.e. that 125I-TC-ricin is transported more rapidly to a later, denser compartment than 125I-TC-AOM) the cells were incubated at 18 degreesC to allow a slower internalisation/transport of the labelled probes. The results obtained showed, again, that the early endosomes containing 125I-TC-ricin were significantly denser than those containing 125I-TC-AOM. We also employed the horseradish peroxidase (HRP)-diaminobenzidine (DAB) density shift technique of Courtoy et al. [6] to determine whether 125I-TC-ricin and 125I-TC-AOM were in separate endosomes early after their uptake. The results showed that early endosomes containing 125I-TC-AOM were density shifted whereas those containing 125I-TC-ricin were unaffected by the density shift procedure. The use of probes labelled with 125I-TC allowed us to identify compartments involved in the degradation of 125I-TC-AOM and 125I-TC-ricin, by measuring acid soluble radioactivities in the gradient fractions. It was found that 125I-TC-ricin was degraded mainly in endosomes, whereas 125I-TC-AOM, as expected, was degraded mainly in lysosomes.

Animals↗
PubMed1998

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells.

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.

Acetylation↗
PubMed1997

[Proteoglycans and pathology--new aspects].

It is well documented that proteoglycans are involved in a wide range of pathological conditions. Recently published results in international journals provide new information on the role of proteoglycans in such conditions. A mutation in the gene encoding for a cell surface proteoglycan has been demonstrated in overgrowth syndromes. A proteoglycan has been isolated from urine and shown to induce cachexia in cancer patients. Furthermore, in both achondrogenesis and colon cancer, the reduced ability to sulphate proteoglycans is due to genetic defects in cellular sulfate transporters. Finally, fibrosis has been inhibited in glomerulonephritic mice by transferring the gene for decorin, a transforming growth factor beta-1 binding proteoglycan, into muscle tissue.

Animals↗
PubMed1997

Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells.

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the basolateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.

Amino Acid Sequence↗
PubMed1996

Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells.

Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.

Amino Acid Sequence↗
PubMed1995

Proteoglycans in polarized epithelial Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

Animals↗