RESUMO
Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.
Assuntos
Cálcio , Mucina-1 , Canais de Cátion TRPV , Animais , Feminino , Humanos , Camundongos , Cálcio/sangue , Cálcio/metabolismo , Cálcio/urina , Membrana Celular/metabolismo , Células Cultivadas , Mucina-1/genética , Mucina-1/metabolismo , Canais de Cátion TRPV/metabolismo , Células Epiteliais/metabolismo , Fatores Sexuais , Mutação , Transporte Proteico/genéticaRESUMO
Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are a disintegrin and metalloprotease 17 (ADAM17) substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 knockout (KO) mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1, and ADAM17 levels (and signaling pathways) were assessed by immunoblot analysis. PT localization was assessed by confocal microscopy and an in situ proximity ligation assay. Findings were extended using human kidneys and urine as well as KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and coexpression of KIM-1 and MUC1 in the PT. Compared with WT mice, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in the PT of WT kidneys but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures compared with WT cultures, whereas inflammation was increased in Muc1 KO kidneys compared with WT kidneys. MUC1 was cleaved by ADAM17 in PT cultures and blocked KIM-1 shedding in Madin-Darby canine kidney cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.NEW & NOTEWORTHY KIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides a decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from experiments in mice, patients, and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.
Assuntos
Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Inflamação/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/irrigação sanguínea , Mucina-1/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteína ADAM17/metabolismo , Animais , Linhagem Celular , Cães , Humanos , Rim/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Mucina-1/genética , Fagocitose/fisiologiaRESUMO
Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.
Assuntos
Arildialquilfosfatase/metabolismo , Canais Epiteliais de Sódio/metabolismo , Adulto , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Canais Epiteliais de Sódio/química , Evolução Molecular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Túbulos Renais Distais/metabolismo , Camundongos , Oócitos/metabolismo , Subunidades Proteicas/metabolismo , RatosRESUMO
The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, ß, and γ). We previously reported that cytoplasmic residues on the ß (ßCys-43 and ßCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over ß subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ß subunit palmitoylation sites. ß subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ß and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.
Assuntos
Aciltransferases/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico/fisiologia , Rim/metabolismo , Processamento de Proteína Pós-Traducional , Aciltransferases/genética , Animais , Células Cultivadas , Citoplasma/metabolismo , Canais Epiteliais de Sódio/genética , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Transporte de Íons , Rim/citologia , Lipoilação , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismo , Subunidades Proteicas , Serina C-Palmitoiltransferase/metabolismo , Sódio/metabolismo , Xenopus laevisRESUMO
Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αßγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, ß-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αßγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+. The lack of N-linked glycans on the ß-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the ß-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type ß-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the ß-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Processamento de Proteína Pós-Traducional , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Glicosilação , Mecanotransdução Celular , Potenciais da Membrana , Mutação , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Tripsina/metabolismo , Xenopus laevisRESUMO
The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and ß strands. Deletion of the peripheral knuckle domain of the α subunit in the αßγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the ß or γ subunit knuckle domain within the αßγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the ß or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.
Assuntos
Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Ratos , Sódio/farmacologiaRESUMO
The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.
Assuntos
Mucina-1/metabolismo , Traumatismo por Reperfusão/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclina D1/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Survivina , Fator de Transcrição 4 , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, ß, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the ß subunit, ßCys-43 and ßCys-557, are Cys-palmitoylated. ENaCs with mutant ßC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the ß and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over ß subunit palmitoylation in modulating ENaC gating.
Assuntos
Cisteína/metabolismo , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Lipoilação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Animais , Sítios de Ligação , Citoplasma/metabolismo , Cães , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos , Subunidades Proteicas/antagonistas & inibidores , Sódio/farmacologiaRESUMO
Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.
Assuntos
Mucina-1/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/fisiopatologiaRESUMO
The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111-1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [(35)S]Met/Cys or glycans with [(3)H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [(3)H]MUC1 when compared with [(35)S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1).
Assuntos
Endossomos/metabolismo , Mucina-1/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cães , Glicosilação , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Mucina-1/química , Mucina-1/genética , Mutação , Transporte ProteicoRESUMO
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.
Assuntos
Polaridade Celular/fisiologia , Mucina-1/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cães , Glicosilação , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Mucina-1/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
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 â 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.
Assuntos
Galectinas/biossíntese , Galectinas/genética , Regulação da Expressão Gênica/fisiologia , Animais , Linhagem Celular , Cães , Galectinas/química , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aßγ channels exhibited increased currents compared with αT663ßγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aßγ and αT663/C664Mßγ channels. The decrease in functional expression of αT663/C664Mßγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.
Assuntos
Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/fisiologia , Mutação/genética , Sequência de Aminoácidos , Animais , Feminino , Humanos , Camundongos , Modelos Animais , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ratos , Transfecção , Xenopus laevisRESUMO
Galectins are best known for their ability to bind glycoconjugates containing ß-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for the preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.
Assuntos
Galectinas , Animais , Carboidratos , Clonagem Molecular , Cães , Galactose/metabolismo , Galectinas/metabolismo , Mamíferos/genéticaRESUMO
The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (α, ß, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the ß and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant ß subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a ß C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na(+) self-inhibition, and reduced single channel P(o) when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that ß Cys-43 is in proximity to the first transmembrane α helix, whereas ß Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that ß subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico/fisiologia , Lipoilação/fisiologia , Sódio/metabolismo , Amilorida/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Simulação por Computador , Cães , Canais Epiteliais de Sódio/genética , Camundongos , Modelos Moleculares , Mutação , Mutação de Sentido Incorreto , Oócitos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Xenopus laevisRESUMO
Galectins (Gal) are ß-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, ß1-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.
Assuntos
Cílios/fisiologia , Cílios/ultraestrutura , Células Epiteliais/fisiologia , Galectinas/fisiologia , Rim/fisiologia , Cicatrização/fisiologia , Animais , Membrana Celular/fisiologia , Células Cultivadas , Cães , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Galectinas/genética , Humanos , Integrina beta1/fisiologia , Rim/citologia , Rim/ultraestrutura , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Túbulos Renais Proximais/ultraestrutura , Camundongos , Camundongos Knockout , Ligação Proteica/fisiologia , Ratos , SuínosRESUMO
The X-linked disorder Lowe syndrome arises from mutations in OCRL1, a lipid phosphatase that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)). Most patients with Lowe syndrome develop proteinuria very early in life. PIP(2) dynamics are known to modulate numerous steps in membrane trafficking, and it has been proposed that OCRL1 activity regulates the biogenesis or trafficking of the multiligand receptor megalin. To examine this possibility, we investigated the effects of siRNA-mediated OCRL1 knockdown on biosynthetic and postendocytic membrane traffic in canine and human renal epithelial cells. Cells depleted of OCRL1 did not have significantly elevated levels of cellular PIP(2) but displayed an increase in actin comets, as previously observed in cultured cells derived from Lowe patients. Using assays to independently quantitate the endocytic trafficking of megalin and of megalin ligands, we could observe no defect in the trafficking or function of megalin upon OCRL1 knockdown. Moreover, apical delivery of a newly synthesized marker protein was unaffected. OCRL1 knockdown did result in a significant increase in secretion of the lysosomal hydrolase cathepsin D, consistent with a role for OCRL1 in membrane trafficking between the trans-Golgi network and endosomes. Together, our studies suggest that OCRL1 does not directly modulate endocytosis or postendocytic membrane traffic and that the renal manifestations observed in Lowe syndrome patients are downstream consequences of the loss of OCRL1 function.
Assuntos
Rim/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Linhagem Celular , Cães , Endocitose/efeitos dos fármacos , Epitélio/metabolismo , Técnicas de Transferência de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Hidrolases/metabolismo , Cinética , Lisossomos/enzimologia , Peso Molecular , Monoéster Fosfórico Hidrolases/genética , Transporte Proteico/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4ß isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4ß, thereby altering its function and that the ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4ß and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4ß altered its lectin binding and secretion. Both ST6GAL1 and MUC4ß inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4ß in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.
Assuntos
Antígenos CD/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inflamação/metabolismo , Mucina-4/metabolismo , Sialiltransferases/metabolismo , Células Th2/imunologia , Adolescente , Adulto , Idoso , Antígenos CD/genética , Antígenos CD/farmacologia , Asma/imunologia , Linhagem Celular , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interleucina-13 , Pulmão , Masculino , Pessoa de Meia-Idade , Mucina-4/genética , Isoformas de Proteínas , Sialiltransferases/genética , Sialiltransferases/farmacologia , Células Th2/efeitos dos fármacos , Transcriptoma , Adulto JovemRESUMO
INTRODUCTION: Plasmin and its precursor, plasminogen, are detectable in urine from patients with glomerular disease. Urinary plasmin(ogen) levels correlate with blood pressure (BP) and may contribute to renal Na+ retention by activating the epithelial Na+ channel (ENaC). In a longitudinal nested-cohort study, we asked whether urinary plasmin(ogen) levels predict subsequent increase in BP, incident hypertension, or mortality in subjects with type I diabetes, who often develop proteinuria. METHODS: The Pittsburgh Epidemiology of Diabetes Complications (EDC) study followed up type I diabetic subjects for 25 years. Urine specimens from 70 subjects with a spectrum of baseline urinary albumin levels were examined. Outcomes included increased BP after 2 years (≥1 SD over baseline systolic or diastolic BP, examined via logistic regression), 25-year incident hypertension (≥140/90 mm Hg or initiating BP-lowering medications), and all-cause or cardiovascular mortality, examined using Cox regression. RESULTS: Subjects experiencing a 2-year increase in BP had higher baseline urinary plasmin(ogen)/creatinine levels (uPl/Cr) than other subjects (P = 0.04); the difference in baseline urinary albumin/creatinine levels (uAlb/Cr) was similar (P = 0.07). Baseline uPl/Cr was associated with increased 25-year hypertension incidence (hazard ratio = 2.05, P = 0.001), all-cause mortality (HR = 2.05, P = 0.01) and cardiovascular mortality (HR = 3.30, P = 0.005), although not independent of uAlb/Cr. CONCLUSION: This is the first long-term prospective study addressing clinical outcomes associated with increased urinary plasmin(ogen). Findings are consistent with a role for plasmin(ogen) in promoting increased BP, but also demonstrate the difficulty in distinguishing effects due to plasmin(ogen) from those of albuminuria.
RESUMO
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.