Paul A. Poland

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.

Recycling of MUC1 Is Dependent on Its Palmitoylation

MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXφ motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.

MUC1 membrane trafficking is modulated by multiple interactions.

MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [35S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr20 and Tyr60 were both required for efficient endocytosis. Mutation of Tyr20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y20HPM is recognized as a YXXϕ motif by the μ2 subunit. The tyrosine-phosphorylated Y60TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr60 blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.

γ‐Glutamyltranspeptidase: Disulfide Bridges, Propeptide Cleavage, and Activation in the Endoplasmic Reticulum

gamma-Glutamyltranspeptidase (gammaGT) is found primarily on the apical surface of epithelial and endothelial cells, where it degrades reduced and oxidized glutathione (gamma-GluCysGly) by hydrolysis of the unique gamma-glutamyl bond. Glutathione plays a key role in disulfide rearrangement in the endoplasmic reticulum (ER) and acts as a redox buffer. Previous work has shown that overexpression of gammaGT or an inactive splice variant gammaGTDelta7 mediates a redox stress response in the endoplasmic reticulum (ER) characterized by increased levels of BiP and induction of CHOP-10. To determine whether a CX(3)C motif might be the common feature of gammaGT and gammaGTDelta7 that mediates this response, we characterized disulfide bridges in gammaGT that might form between the six highly conserved Cys residues. Using site-directed mutagenesis of gammaGT, expression in Chinese Hamster Ovary (CHO) cells, metabolic labeling, and immunoblotting, our data predict disulfide formation between Cys49 and Cys73 and between Cys191 and Cys195 (the CX(3)C motif). Potential functions for this CX(3)C motif are discussed. In the course of defining the disulfides, we also noted that propeptide cleavage correlated with enzymatic activity. Because recent reports indicate that the homologous Escherichia coli gammaGT is a member of the N-terminal nucleophile (Ntn) hydrolase family, where the amino acid at the new N-terminus functions as the nucleophile for both autocatalytic cleavage and enzymatic activity, the rat gammaGT was similarly characterized. As predicted, mutations at the propeptide cleavage site coincidentally inhibit both heterodimer formation and gammaGT enzymatic activity. Analysis of early cleavage events using cell extraction into SDS indicates that propeptide cleavage occurs while gammaGT is still within the ER. Because activation and cleavage are coincident events, this raises the new question of whether an active glutathionase is present within the ER and what role gammaGT plays in modulating ER glutathione levels that are so critical for proper redox balance and disulfide formation in this compartment.

Small Heat Shock Protein αA-crystallin Regulates Epithelial Sodium Channel Expression

Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein αA-crystallin, have recently been shown to play a role in this process. We have now found that αA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na+ transport is mediated by epithelial Na+ channels (ENaC). ENaC-mediated Na+ currents in Xenopus oocytes were reduced by co-expression of αA-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of αA-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that αA-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation.

Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells

Madin Darby canine kidney (MDCK) cells are a well characterized epithelial cell line used to study mechanisms of polarized delivery. As glycans on apically expressed proteins have been identified as targeting signals, and crosslinking by the abundant galectin-3 has been implicated in the mechanism of glycan-dependent sorting, we wanted to identify other members of the galectin (Gal) family expressed in MDCK cells. By analyzing intron-exon boundaries, we identified canine genes that were highly homologous to mammalian Gal-1, 2, 3, 4, 7, 8, 9, and 12, and galectin-related HSPC159 and GRIFIN. Transcripts for Gal-2 and -12 were not detected in MDCK cells, but we found transcript levels for Gal-3 > Gal-9 > Gal-8 > Gal-1 ⋙ Gal-4 > Gal-7. Canine Gal-1, -2, -3, -4, -7, -8, -9, and -12 were cloned and expressed in Escherichia coli as GST fusion proteins to characterize binding specificities on arrays of synthetic glycans on glass slides from Core H of the NIH Consortium for Functional Glycomics. Individual expression of the N-terminal (GST-Gal-9N) and C-terminal (GST-Gal-9C) carbohydrate recognition domains greatly improved protein yield and the ability to characterize Gal-9 binding on the array. Canine galectins differentially bound sulfated disaccharides as well as human blood groups A, B, and H on both N-glycans and linear glycan structures on the array. Analysis of GST-Gal-1, -3, -4, -7, -8, -9N, and -9C binding to immunopurified human MUC1 expressed in MDCK cells revealed a preference for binding GST-Gal-3 and -9, which interestingly reflects the two most abundant galectins expressed in MDCK cells.

Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells.

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

Core-glycosylated mucin-like repeats from MUC1 are an apical targeting signal.

Background: MUC1 apical delivery in polarized MDCK cells employs a path used by proteins with glycan-dependent targeting signals. Results: Core O-glycans on mucin-like repeats of MUC1 act as an apical targeting signal. Conclusion: MUC1 apical targeting is O-glycan-dependent. Significance: We have identified a specific sequence with a post-translational modification that can direct apical delivery of MUC1 or a reporter protein. MUC1 is efficiently delivered to the apical surface of polarized Madin-Darby canine kidney (MDCK) cells by transit through apical recycling endosomes, a route associated with delivery of apical proteins with glycan-dependent targeting signals. However, a role for glycans in MUC1 sorting has not been established. A key feature of MUC1 is a heavily O-glycosylated mucin-like domain with a variable number of nearly perfect tandem repeats and adjacent imperfect repeats. Metabolic labeling, cell surface biotinylation, immobilized lectins, and confocal immunofluorescence microscopy were used to characterize the polarized delivery of MUC1 mutants and chimeras in MDCK cells to identify the apical targeting signal. Both the interleukin-2 receptor α subunit (Tac) and a chimera where the Tac ectodomain replaced that of MUC1 were delivered primarily to the basolateral surface. Attachment of the MUC1 mucin-like domain to the N terminus of Tac enhanced apical but not basolateral delivery when compared with Tac. Conversely, deletions within the mucin-like domain in MUC1 reduced apical but not basolateral delivery when compared with MUC1. In pull-down assays with lectins, we found a notable difference in the presence of core 1 O-glycans, but not poly-N-acetyllactosamine, in apically targeted MUC1 and chimeras when compared with Tac. Consistent with these data, we found no effect on MUC1 targeting when galectin-3, with preference for poly-N-acetyllactosamine, was depleted from polarized MDCK cells. However, we did block the apical targeting activity of the mucin-like repeats when we overexpressed CMP-Neu5Ac:GalNAc-Rα2,6-sialyltransferase-1 to block core O-glycan synthesis. The cumulative data indicate that the core-glycosylated mucin-like repeats of MUC1 constitute an apical targeting signal.

Differential glycosylation of MUC1 in tumors and transfected epithelial and lymphoblastoid cell lines

The membrane-bound mucin-like protein MUC1 with a specified number of tandem repeats has been expressed by transfection of the cDNAs in both the epithelial cell lines MDCK and LLC-PK1, and human lymphoblastoid cell lines T2 and C1R. The structure and glycosylation states of the MUC1 in these four lines were compared with that of the endogenous MUC1 found in the human pancreatic (HPAF) and breast (BT-20) tumor cell lines using flow cytometry and Western blot analysis with anti-MUC1 antibodies, which are either sensitive or insensitive to the glycosylation state of the tandem repeat, and pretreatment of cells with phenyl-α-galactosaminide, an inhibitor of mucin sialylation. A similar analysis of MUC1 expression in transfected normal and O-glycosylation defective CHO cells reveals that the addition of galactose to the core oligosaccharide structure is apparently responsible for the anomalous difference in Mr between the mature and propeptide forms of the MUC1. Both the tumor cells and the transfected lymphoblastoid cells consistently express significant steady state levels of both the heavily glycosylated mature forms and the poorly glycosylated propeptide forms of the MUC1, whereas MUC1 is found predominantly as the mature extensively glycosylated species in the transfected epithelial cells. Immunofluoresence microscopy of cross sections of the polarized epithelial cells grown on culture filter inserts reveals that the MUC1 is clearly present at the apical surface of the cells, consistent with its expression in normal tissues. Thus, the successful expression of the MUC1 by transfection of either lymphoblastoid cells or epithelial cells yields model systems both for studying the natural structure/function relationships of the protein domains within the MUC1 molecule and for further elucidating the previously reported MHC-independent T-cell recognition of the MUC1.

Differential expression of MUC1 on transfected cell lines influences its recognition by MUC1 specific T cells

In adenocarcinomas of the breast and pancreas, underglycosylation of the glycoprotein MUC1, also expressed by normal breast and pancreatic ductal epithelial cells, results in new protein epitopes to which the immune system mounts a cytotoxic T cell response. This cytotoxic immune response is directed primarily against epitopes on the tandem repeat domain of MUC1, and is unconventional in that it is major histocompatibility complex (MHC)-unrestricted. It is therefore necessary to investigate the molecular basis of this immune response in order to enhance and optimize it for immune therapy purposes. In the present study, we characterize new MUC1 transfected human lymphoblastoid cell lines C1R and T2, and a pig kidney epithelial line LLC-PK1, that express MUC1 with either two repeats (MUC1–2R) or 22 repeats (MUC1–22R), and use them as stimulators and targets for cytotoxic T cells (CTL)in vitro. We show that MUC1–2R is processed and glycosylated similarly to MUC1–22R. In contrast to MUC1–22R, MUC1–2R is not recognized by CTL on T2 and C1R cells known for no or low MHC class I expression. It is however recognized when expressed at high density on xenogeneic LLC-PK1 cells. We propose that in MHC-unrestricted recognition, a large number of MUC1 epitopes is necessary to effectively engage the T cell receptor, and that in the presence of a low number of epitopes, engagement of the CD8 co-receptor by MHC class I molecules may be required for completing the signal through the T cell receptor.