Secretory Glycoprotein Research Articles

Abstract #128: CA-125 Glycoprotein Secretion by an Androgen-Secreting Adrenal Adenoma in an Adult Female Patient

Abstract #128: CA-125 Glycoprotein Secretion by an Androgen-Secreting Adrenal Adenoma in an Adult Female Patient

Open-channel microfluidic membrane device for long-term FT-IR spectromicroscopy of live adherent cells.

Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air-liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems.

Prognostic value of tissue inhibitor of metalloproteinase-2 expression in patients with non-small cell lung cancer: a systematic review and meta-analysis.

Background and ObjectivesTissue inhibitor of metalloproteinase-2 (TIMP-2) is a small secretory glycoprotein with anti–matrix metalloproteinase activity. Data on the value of TIMP-2 as a prognostic factor in non–small cell lung cancer (NSCLC) are discordant and remain controversial. A systematic review and meta-analysis was performed to explore this issue.MethodsWe identified the relevant literature by searching the PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure, SinoMed, and Wanfang Data databases (search terms: “non-small cell lung cancer” or “NSCLC” or “Lung Carcinoma, Non-Small-Cell”, “Tissue Inhibitor of Metalloproteinase-2” or “TIMP-2”, and “prognosis” or “prognostic” or “survive”) for updates prior to March 1, 2014. The pooled hazard ratio (HR) of overall survival with a 95% confidence interval (95% CI) was used to evaluate the strength of the association between positive TIMP-2 expression and survival in patients with NSCLC.ResultsWe included 12 studies in our systematic review; five studies involving 399 patients with NSCLC were meta-analyzed. The pooled HR of all included patients was 0.57 (95% CI: 0.43–0.77), and the HRs of subgroup analysis according to stage (I–IV), testing method (immunohistochemistry) and high TIMP-2 expression percentage (<50%) were 0.63 (95% CI: 0.43–0.92), 0.55 (95% CI: 0.41–0.74), and 0.50 (95% CI: 0.28–0.88), respectively. These data suggested that high TIMP-2 expression is associated with favorable prognosis in NSCLC. The meta-analysis did not reveal heterogeneity or publication bias.ConclusionsTIMP-2 expression indicates favorable prognosis in patients with NSCLC; as a protective factor, it could help predict outcome and may guide clinical therapy in the future.

T3 rapidly regulates several steps of alpha subunit glycoprotein (CGA) synthesis and secretion in the pituitary of male rats: Potential repercussions on TSH, FSH and LH secretion

TSH, FSH and LH share the same glycoprotein alpha chain (CGA) as part of their protein structure. Therefore, it is possible that thyroid and gonadal dysfunction may affect the CGA expression. This study evaluated several steps of CGA synthesis and secretion in thyrotrophs and gonadotrophs of control and hypothyroid rats, acutely or chronically-treated with T3. Hypothyroidism increased the Cga mRNA expression and its association to ribosome, but decreased intracellular CGA content. These parameters were reversed after acute or chronic T3 treatment. We conclude that T3 not only down-regulates Cga mRNA expression, as expected, but also inhibits the association of Cga mRNA to ribosome, as well as the CGA secretion. These findings add novel insights into our understanding of the role of T3 on the regulation of the Cga gene expression and CGA secretion, which might have a potential repercussion in all pituitary glycoprotein hormone synthesis and secretion.

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

ER Stress During the Pubertal Growth Spurt Results in Impaired Long-Bone Growth in Chondrocyte-Specific ERp57 Knockout Mice.

Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.

Light microscopy and scanning electron microscopy study on microstructure of gallbladder mucosa in pig.

The present light microscopy (LM) and scanning electron microscopy (SEM) studies on porcine gallbladder mucosa provide a description of the microstructures of great functional importance such as mucosal folds, the epithelium, glands, and lymphatic nodules. The results showed the regional structural differences of the porcine gallbladder wall. Depending on the part of the gallbladder, three types of mucosal structures were described: simple and branched folds and mucosal crypts. An important structural feature found in the mucosa is connected with the structural variety of type of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in the polygonal crypts toward the fundus of the gallbladder. The morphometric analysis showed statistically significantly differences in the form and size of the folds and between the fundus, body, and neck of the gallbladder. Differences in the size of mucosal epithelium are discussed in terms of processes of synthesis and secretion of glycoproteins. Regional, species-specific differences in morphology of mucosal subepithelial glands, i.e., their secretory units and openings, and intensity of mucus secretion were described. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the mucosa for (1) secretion of mucus in the neck or body of gallbladder and (2) for cyclic volume changes, especially in the fundus of gallbladder. The description of the microstructures of mucosa in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation.

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.

Association of malectin with ribophorin I is crucial for attenuation of misfolded glycoprotein secretion

We previously demonstrated that malectin associates with ribophorin I, which is a subunit of oligosaccharyltransferase in the endoplasmic reticulum (ER). When malectin and ribophorin I are overexpressed in the ER, secretion of an α1-antitrypsin (AT) variant whose folding is defective, termed null Hong Kong (ATNHK), is decreased. To confirm whether the interaction between malectin and ribophorin I is involved in the decreased secretion of misfolded glycoproteins, we constructed an expression vector encoding truncated malectin, which could not associate with ribophorin I and had an Lys-Asp-Glu-Leu ER-retention sequence at its C-terminus. Expression of wild-type malectin abrogated ATNHK secretion, whereas expression of truncated malectin did not affect ATNHK secretion. Both wild-type and truncated malectin bound to ATNHK, and the level of ATNHK was similar in cells expressing wild-type malectin and those expressing truncated malectin. Furthermore, we previously showed that decreased secretion of misfolded ATNHK by malectin overexpression is restored by treatment with a proteasome inhibitor. These results clearly demonstrate that the association of malectin with ribophorin I is required to capture misfolded glycoproteins and direct them to the degradation pathway.

TNFRSF11B gene polymorphisms increased risk of peripheral arterial occlusive disease and critical limb ischemia in patients with type 2 diabetes.

Osteoprotegerin (OPG) is a secretory glycoprotein that belongs to the tumor necrosis factor receptor family and plays a role in atherosclerosis. OPG has been hypothesized to modulate vascular functions; however, its role in mediating atherosclerosis is controversial. Epidemiological data in patients with cardiovascular disease (CVD) indicate that OPG serum levels are associated with several inflammatory markers, myocardial infarction events, and calcium scores, suggesting that OPG may be causative for CVD. The present study aimed to evaluate whether the OPG gene (TNFRSF11B) polymorphisms are involved in the development of peripheral arterial occlusive disease (PAOD) and critical limb ischemia (CLI) in patients with type 2 diabetes. This genetic association study included 402 diabetic patients (139 males and 263 females) with peripheral arterial occlusive disease and 567 diabetic subjects without peripheral arterial occlusive disease (208 males and 359 females). The T245G, T950C, and G1181C polymorphisms of the OPG gene were analyzed by polymerase chain reaction and restriction fragment length polymorphism. We found that the T245G, T950C, and G1181C gene polymorphisms of the OPG gene were significantly (27.9 vs. 12.2%, P<0.01; 33.6 vs. 10.4%, P<0.01 and 24.4 vs. 12.7%, P<0.01, respectively) and independently (adjusted OR 4.97 (3.12-6.91), OR 7.02 (4.96-11.67), and OR 2.85 (1.95-4.02), respectively) associated with PAOD. We also found that these three polymorphisms act synergistically in patients with PAOD and are associated with different levels of risk for PAOD and CLI, depending on the number of high-risk genotypes carried concomitantly by a given individual. The TNFRSF11B gene polymorphisms under study are associated with PAOD, and synergistic effects between these genotypes might be potential markers for the presence and severity of atherosclerotic disorders.

Deletion of olfactomedin 2 induces changes in the AMPA receptor complex and impairs visual, olfactory, and motor functions in mice

Olfactomedin 2 (Olfm2) is a secretory glycoprotein belonging to the family of olfactomedin domain-containing proteins. A previous study has shown that a mutation in OLFM2 is associated with primary open angle glaucoma in Japanese patients. In the present study, we generated Olfm2 deficient mice by replacing the Olfm2 gene with the LacZ gene. The loss of Olfm2 resulted in no gross abnormalities. However, Olfm2 null mice showed reduced exploration, locomotion, olfactory sensitivity, abnormal motor coordination, and anxiety related behavior. The pattern of the Olfm2 gene expression was studied in the brain and eye using β-galactosidase staining. In the brain, Olfm2 was mainly expressed in the olfactory bulb, cortex, piriform cortex, olfactory trabeculae, and inferior and superior colliculus. In the eye expression was detected mainly in retinal ganglion cells. In Olfm2 null mice, the amplitude of the first negative wave in the visual evoked potential test was significantly reduced as compared with wild-type littermates. Olfm2, similar to Olfm1, interacted with the GluR2 subunit of the AMPAR complexes and Olfm2 co-segregated with the AMPA receptor subunit GluR2 and other synaptic proteins in the synaptosomal membrane fraction upon biochemical fractionation of the adult mice cortex and retina. Immunoprecipitation from the synaptosomal membrane fraction of the Olfm2 null mouse brain cortex using the GluR2 antibody showed reduced levels of several components of the AMPAR complex in the immunoprecipitates including Olfm1, PSD95 and CNIH2. These results suggest that heterodimers of Olfm1 and Olfm2 interact with AMPAR more efficiently than Olfm2 homodimers and that Olfm2 plays a role in the organization of the AMPA receptor complexes.

Glycosylation and immunoreactivity of the Histoplasma capsulatum Cfp4 yeast-phase exoantigen.

The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.

Chronic aluminum intoxication in rat induced both serotonin changes in the dorsal raphe nucleus and alteration of glycoprotein secretion in the subcommissural organ: Immunohistochemical study

Aluminum (Al) causes multiple impairments in several body systems including the central nervous system. In fact, Al exposure has been mostly associated with neurological dysfunctions that occur in some brain diseases. The effect of Al neurotoxicity on the dopaminergic system is well documented, but this effect on the serotoninergic system is poorly studied. The aim of this work is to evaluate the effect of chronic Al intoxication (0.3% of aluminum chloride exposure from the intra-uterine age until 4 months of adult age) on dorsal raphe nucleus (DRN) which is the main source of serotonin, and also on the glycoprotein secretion of subcomissural organ (SCO), receiving important serotoninergic innervation. This will be executed using immunohistochemistry procedure, with both the anti serotonin and the anti Reissner's fiber antibodies in the rat. Our results showed a significant increase of serotonin immunoreactivity in the DRN, accompanied by a noticeable decrease of RF immunoreactivity in the SCO ependymocytes. This study provides further evidence confirming the toxic effect of Al exposure on serotonin neurotransmission in the brain likely through increased synthesis or decreased release. Al exposure was also shown to decrease RF glycoprotein which is involved in the detoxification of cerebrospinal fluid.

Sex-response differences of immunological and histopathological biomarkers in gill of Prochilodus argenteus from a polluted river in southeast Brazil

The fish gill is in direct and standing contact with the immediate external environment and, therefore, is highly vulnerable to aquatic pollutants. In this study, Prochilodus argenteus were caught at two different points in São Francisco river. The first point is located near Três Marias dam, while the second is placed downstream the Abaeté river. Chemical approaches showed the presence of metals contamination in the first point. Thus, the main goal of this study was to investigate the possible toxic effects of these contaminants and the likely use of biomarkers on fish gills. Biometric data of length and weight of fish were obtained in order to calculate the condition factor as an organismal biomarker. The histological changes in gills and alterations in mucous and rodlet cells occurrence were detected microscopically and evaluated with quantitative analyses. Myeloperoxidase (MPO) and Eosinophil Peroxidase (EPO) were also assessed in fish gill. The analysis of the water and sediment samples revealed the presence of metals at the two points. As and Cd were detected at higher concentrations at point 1. The presence of lamellar cell hyperplasia, lamellar fusion, lamellar edema and inflammatory foci varied according to the point. Additionally, mucous and rodlet cells and MPO and EPO activities showed variability according to the environmental conditions. Furthermore, with exception of lamellar hyperplasia and eosinophil peroxidase activity, all others parameters showed sex-variation responses. At the first point, male fish showed a chronical inflammation in gills due to the lowest activity of MPO and EPO, as well as low occurrence of inflammatory foci and glycoprotein secretion by mucous cells, while female fish presented an opposite pattern of response to the same environmental conditions. Therefore, we suggest the use of such biomarkers in future monitoring of aquatic systems, taking into account the sex-variation responses.

Leukotriene enhanced allergic lung inflammation through induction of chemokine production (HYP4P.414)

Abstract Leukotrienes, which are mainly expressed by mast cells, macrophages and eosinophils, have been shown to act as important mediators in allergic asthma. In addition to leukotrienes, IL-13, a Th2 specific cytokine, has also been considered as the most important effector cytokine to induce allergic asthma, by increasing airway hyper-responsiveness, airway inflammation, eosinophilia and mucus hyper-production. But, the exact mechanisms how leukotrienes elicit allergic lung responses at initial stage and the effect of leukotrienes on allergic asthma through IL-13 remain unclear. To reveal this, we adopted two distinct models of allergic inflammation: intranasal Aspergillus protease allergen and recombinant IL-13 induced Th2 mediated inflammation. Here we measured AHR, bronchoalveolar lavage cytology, glycoprotein secretion, as well as IL-13 related chemokines such as CCL7, CCL11 and CCL17, which directly regulate homing of Th2 cells to lung with 5-lipoxygenase deficient and wild type mice. 5-lipoxygenase that convert arachidonic acid into leukotriene A4 null mice showed attenuated airway inflammation, AHR and bronchoalveolar lavage eosinophilia, as well as diminished chemokines including CCL7 and CCL17. Finally, addition of LTB4 and LTC4 to the airways of 5-lipoxygenase deficient mice restored rIL-13 induced asthma. Thus, our results indicate that LTs augment the effect of IL-13 in allergic asthma, are required for Th2 specific chemokine production.

Effect of NADPH oxidase 2 (Nox2) deficiency on the function of Th2 cells (HYP4P.415)

Abstract Nox2/gp91phox, one of phagocyte NADPH oxidase subunits, is mainly expressed on phagocytes. They transport electrons across the plasma membranes and kill pathogens by producing ROS. Many evidences have shown that Nox2 deficiency causes susceptibility to infections and immunosuppression. Recent studies have identified functional phagocyte-type Nox2 is also expressed in helper T cells through TCR stimulation. So, many papers attempted to define the role of Nox2 on Th1 and Th17 cell differentiation, but the exact function of Nox2 in Th2 differentiation remains unclear. To elucidate this, we adopted experimental allergic asthma that Th2 cells have been considered as a pivotal mediator for asthma pathogenesis. Here, we revealed that Nox2 deficient mice showed enhanced allergic asthma phenotypes including increased airway hyper responsiveness, eosinophilia, glycoprotein secretion and IL-4 production in bronchoalveolar lavage through augmented Th2 differentiation, regardless of challenge routes and allergen types. Based on our results, we conclude that Nox2 could be a molecular target for controlling Th2 cell differentiation and allergic asthma.

Prognostic implications of SLIT and ROBO1 expression in gallbladder cancer.

SLIT, a secretory glycoprotein, and its receptor roundabout (ROBO) are expressed in several types of cancer and have been implicated in tumor angiogenesis. The purpose of this study was to determine the prognostic implications of SLIT and ROBO1 expression and their association with clinicopathologic characteristics in gallbladder cancer. A retrospective analysis of 109 consecutive patients who underwent primary gallbladder cancer resection was conducted. Univariate and multivariate models were used to analyze the effect of clinicopathologic factors on survival. Expression of SLIT and ROBO1 was evaluated by immunohistochemistry, and their association with clinicopathologic characteristics was analyzed using mean testing. Multivariate linear regression analysis was also applied to investigate the independent predictors of ROBO1 expression. Seventy-five patients were included in the post-resection survival analysis, with 1-year and 3-year overall survival rates of 60 and 40 %, respectively. Univariate analysis revealed that pN classification, pT classification, pM classification, liver involvement, perineural invasion, TNM staging, Nevin staging, and microscopic resection margins affect prognosis. Multivariate analysis confirmed that pN classification, liver involvement, and perineural invasion are independent prognostic factors. In the mean tests of 109 cases, the mean difference of SLIT immunoreactivity was significant according to the presence of gallstones (P = 0.003) and liver involvement (P = 0.005), while the mean difference of ROBO1 immunoreactivity was significant according to liver involvement (P < 0.001), TNM staging (P < 0.001), and Nevin staging (P < 0.001). Multivariate analysis of ROBO1 immunoreactivity showed that SLIT immunoreactivity and TNM stage (adjusted R (2) = 0.203) or SLIT immunoreactivity and Nevin stage (adjusted R (2) = 0.195) were independent predictors of ROBO1 expression. pN classification, liver involvement, perineural invasion, and pathologic stage are significant prognostic factors for gallbladder cancer survival. SLIT expression is associated with cholelithiasis and liver involvement, and ROBO1 expression is associated with liver involvement and pathologic stage. In addition, SLIT expression and pathologic stage predict ROBO1 expression independently.

Characterization of V-ATPase inhibitor-induced secretion of cysteine-rich with EGF-like domains 2

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure. The levels of secretion of EGFP-fused CRELD2 (SP-EGFP-CRELD2), which consists of EGFP following the putative signal peptide (SP) sequence of CRELD2, from COS7 cells transiently transfected with this construct were also increased after each of the treatments, but their intracellular localization was barely affected by CMA treatment. Transient overexpression of 78-kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) also increased the secretion of CRELD2 from HEK293 cells expressing wt CRELD2, whereas the perturbation of intravesicular acidification did not alter the expression of GRP78 and PDI in the HEK293 cells. We further studied the roles of intracellular calcium ions and the Golgi apparatus in the secretion of CRELD2 from HEK293 cells in which intravesicular acidification was perturbed. The treatment with calcium ionophore increased the secretion of wt CRELD2, while that with BAPTA-AM, an intracellular calcium chelator, did not reduce the CMA-induced CRELD2 secretion. By contrast, treatment with brefeldin A (BFA), which inhibits the transportation of proteins from the ER to the Golgi apparatus, almost completely abolished the secretion of wt CRELD2 from the HEK293 cells. In conclusion, we demonstrated that the intravesicular acidification by V-ATPase regulates the secretion of CRELD2 without relying on the balance of intracellular calcium ions and the expression of ER chaperones such as GRP78 and PDI. These findings concerning the role of V-ATPases in modulating the secretion of CRELD2, a novel ER stress-inducible secretory factor, may provide new insights into the prevention and treatment of certain ER stress-related diseases.

Oligomerization-induced Conformational Change in the C-terminal Region of Nel-like Molecule 1 (NELL1) Protein Is Necessary for the Efficient Mediation of Murine MC3T3-E1 Cell Adhesion and Spreading

NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin 1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine-mutant) from a mammalian expression system and tested their activities. A deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for the cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary but that other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1, which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1.