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The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.
Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.
Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.
We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[(3)H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.
ATF6 is a membrane-bound transcription factor that activates genes in the endoplasmic reticulum (ER) stress response. When unfolded proteins accumulate in the ER, ATF6 is cleaved to release its cytoplasmic domain, which enters the nucleus. Here, we show that ATF6 is processed by Site-1 protease (S1P) and Site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation. ATF6 processing was blocked completely in cells lacking S2P and partially in cells lacking S1P. ATF6 processing required the RxxL and asparagine/proline motifs, known requirements for S1P and S2P processing, respectively. Cells lacking S2P failed to induce GRP78, an ATF6 target, in response to ER stress. ATF6 processing did not require SCAP, which is essential for SREBP processing. We conclude that S1P and S2P are required for the ER stress response as well as for lipid synthesis.
The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: alpha, beta and gamma. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications.
Antibodies have been raised against synthetic peptides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitive norepinephrine (NE) transporter (NET). One antibody (N430), raised and purified against a putative intracellular human norepinephrine transporter (hNET) epitope, detects hNET expression in a stably transfected cell line (LLC-NET) by indirect immunofluorescence only in the presence of detergent, while no immunoreactivity is observed in either the parental cells (LLC-PK1) or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species in these cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparent M(r) of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA. PNGase F digestion of extracts prepared from LLC-NET- and hNET-transfected HeLa cells convert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosylation inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologically similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does not contribute significantly to antagonist sensitivity. On the other hand, NE transport and antagonist ([125I]RTI-55) binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an inability of NET proteins to replenish the higher M(r) hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosynthetic maturation, stability, and functional expression. In summary, N430 antibody is a useful tool for the visualization and characterization of hNET gene products and has permitted the first direct evaluation of biosynthetic steps leading to functional catecholamine transporter expression.
Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC* assays. MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.
Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. A locus, v-erbB, within the viral genome has been implicated in AEV-mediated oncogenesis. We report here the detection and partial characterization of the protein product of the v-erbB oncogene in AEV-transformed cells. We obtained the antisera necessary for our analysis by expressing a portion of the molecularly cloned v-erbB locus in Escherichia coli and immunizing rabbits with the resulting bacterial erbB polypeptide. Antisera directed against the bacterial polypeptide reacted with v-erbB proteins obtained from virus-infected avian cells. By three criteria--tunicamycin inhibition, lectin binding and metabolic labeling with radioactive sugar precursors--the product of the v-erbB gene appears to be a glycoprotein.
The effect of glycosylation on the assembly and antigenicity of HLA antigens was investigated by examining HLA antigens synthesized in the presence of the antibiotic tunicamycin, an inhibitor of asparagine-linked oligosaccharide addition, with monoclonal antibodies specific for a variety of antigenic determinants. The monoclonal antibody Q5/13 reactive with a determinant expressed on the beta chain of human Ia-like antigens immunoprecipitated alpha and beta subunits with reduced apparent molecular weights from tunicamycin-treated cells, indicating that glycosylation is not required for association of the Ia-like antigen alpha and beta subunits. Immunoprecipitation of HLA-A,B,C antigens from tunicamycin-treated cells with four monoclonal antibodies specific for the heavy chain and one specific for beta 2-microglobulin showed that the heavy-chain determinant detected by the antibody Q6/64 is absent from the non-glycosylated molecule. This is the first demonstration that carbohydrate addition during biosynthesis affects the protein conformation of the HLA-A,B,C heavy chain.