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Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells.

ABSTRACT: Filter-grown Madin-Darby canine kidney (MDCK) cells labelled for 24 h with [35S]sulphate were found to secrete macromolecules [35S]sulphated on their carbohydrate moieties predominantly into the basolateral medium, whereas the tyrosine-[35S]sulphated proteins synthesized were predominantly secreted into the apical medium. In contrast with the predominant apical secretin of tyrosine-[35S]sulphated proteins, the free tyrosine O-[35S]sulphate (Tyr[35S]) was released mostly into the basolateral medium. A time-lapse study using prelabelled MDCK cells incubated in fresh medium revealed that, during the 48 h time course monitored, the release of tyrosine-[35S]sulphated proteins into the apical medium was faster and quantitatively greater than that into the basolateral medium. During the same time there was a concomitant release, predominantly into the basolateral medium, of the free Tyr[35S] derived from the degradation of tyrosine-[35S]sulphated proteins. An endocytotic degradation experiment was performed to demonstrate the endocytosis of tyrosine-sulphated proteins and their degradation to generate free TyrS. It was found that free Tyr[35S] was generated and released when an apically secreted (or basolaterally secreted) tyrosine-[35S]sulphated protein preparation was added to the apical medium (or the basolateral medium) of unlabelled filter-grown MDCK cells. In both cases, the free Tyr[35S] generated was predominantly released into the basolateral medium similar to the results obtained in the time-lapse study.

SUBMITTER: Han JR

PROVIDER: S-EPMC1151578 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Project description: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.

Project description:By employing an affinity-gel fractionation technique coupled to Western-blot analysis, we have identified a 175 kDa tyrosine-O-sulphate (TyrS)-binding protein present in Madin-Darby canine kidney (MDCK) cells. The binding of this TyrS-binding protein to TyrS covalently bonded to Sepharose gel was found to be pH-dependent, being strong from pH 8.0 down to pH 6.5 and increasingly weak at pH 6.0 and below. Results obtained from Triton X-114 temperature-induced phase separation and sodium carbonate buffer (pH 11) extraction experiments indicated that the TyrS-binding protein is an integral membrane protein. This 175 kDa TyrS-binding protein was found to be present in association with a major tyrosine-sulphated protein, the apically secreted 80 kDa glycoprotein (gp 80), in cell lysate prepared from MDCK cells maintained under normal growth conditions. When the cell lysate used was prepared from MDCK cells pretreated with 20 mM sodium chlorate, a metabolic sulphation inhibitor, the complex formed between the two proteins could no longer be detected, indicating that the binding of the TyrS-binding protein is through the TyrS residue(s) of gp 80. Both cell-surface biotinylation and cell-surface trypsinization studies demonstrated the predominantly, if not exclusively, intracellular location of the TyrS-binding protein. Furthermore, radioactive pulse-chase experiments revealed that the newly synthesized radiolabelled fibronectin and gp 80 were present in complexes with the TyrS-binding protein in MDCK cells pulse-labelled with [35S]methionine or [35S]sulphate. Exogenous [35S]methionine-labelled gp 80 added to the medium, on the other hand, was not found to be present in association with the TyrS-binding protein in MDCK cells over a 2-h time course. These results strongly suggested the identity of the 175 kDa TyrS-binding protein as a putative 'TyrS receptor', possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in MDCK cells.

Project description:Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.

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Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells.

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