Non-glycosylated Membrane Research Articles

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

Hypoxia-dependent expression of MAP17 coordinates the Warburg effect to tumor growth in hepatocellular carcinoma

BackgroundReprogrammed glucose metabolism, also known as the Warburg effect, which is essential for tumor progression, is regarded as a hallmark of cancer. MAP17, a small 17-kDa non-glycosylated membrane protein, is frequently dysregulated in human cancers. However, its role in hepatocellular carcinoma (HCC) remains largely unknown.MethodsImmunohistochemistry was used to analyze the expression pattern of MAP17 in HCC. Loss-of-function and gain-of-function studies were performed to investigate the oncogenic roles of MAP17 in vitro and in vivo. RNA sequencing, co-immunoprecipitation, immunofluorescence and western blotting were used to study the molecular mechanism of MAP17 affecting the tumor growth and glycolytic phenotype of HCC.ResultsAn integrative analysis showed that MAP17, a small 17-kDa non-glycosylated membrane protein, is significantly related to the glycolytic phenotype of hepatocellular carcinoma (HCC). Firstly, we found that MAP17 expression is hypoxia-dependent and predicts a poor prognosis in HCC. Genetic silencing of MAP17 reduced the rate of glucose uptake, lactate release, extracellular acidification rate, and expression of glycolytic genes. Ectopic expression of wild type MAP17 but not its PDZ binding domain mutant MAP17-PDZm increased tumor glycolysis. Further research showed that MAP17 knockdown markedly retarded in vivo tumor growth in HCC. Importantly, attenuation of tumor glycolysis by galactose largely hijacked the growth-promoting role of MAP17 in HCC cells. RNA sequencing analysis revealed that MAP17 knockdown leads to transcriptional changes in the ROS metabolic process, cell surface receptor signaling, cell communication, mitotic cell cycle progression, and regulation of cell differentiation. Mechanistically, MAP17 exerted an increased tumoral phenotype associated with an increase in reactive oxygen species (ROS), which activates downstream effectors AKT and HIF1α to enhance the Warburg effect. In HCC clinical samples, there is a close correlation between MAP17 expression and HIF1α or phosphorated level of AKT.ConclusionsOur results show that MAP17 is a novel glycolytic regulator, and targeting MAP17/ROS pathway may be an alternative approach for the prevention and treatment of HCC.

MAP17 (PDZK1IP1) and pH2AX are potential predictive biomarkers for rectal cancer treatment efficacy.

Rectal cancer represents approximately 10% of cancers worldwide. Preoperative chemoradiotherapy increases complete pathologic response and local control, although it offers a poor advantage in survivorship and sphincter saving compared with that of radiotherapy alone. After preoperative chemoradiotherapy, approximately 20% of patients with rectal cancer achieve a pathologic complete response to the removed surgical specimen; this response may be related to a better prognosis and an improvement in disease-free survival. However, better biomarkers to predict response and new targets are needed to stratify patients and obtain better response rates.MAP17 (PDZK1IP1) is a small, 17 kDa non-glycosylated membrane protein located in the plasma membrane and Golgi apparatus and is overexpressed in a wide variety of human carcinomas. MAP17 has been proposed as a predictive biomarker for reactive oxygen species, ROS, inducing treatments in cervical tumors or laryngeal carcinoma. Due to the increase in ROS, MAP17 is also associated with the marker of DNA damage, phosphoH2AX (pH2AX). In the present manuscript, we examined the values of MAP17 and pH2AX as surrogate biomarkers of the response in rectal tumors. MAP17 expression after preoperative chemoradiotherapy is able to predict the response to chemoradiotherapy, similar to the increase in pH2AX. Furthermore, we explored whether we can identify molecular targeted therapies that could help improve the response of these tumors to radiotherapy. In this sense, we found that the inhibition of DNA damage with olaparib increased the response to radio- and chemotherapy, specifically in tumors with high levels of pH2AX and MAP17.

Elevation of MAP17 enhances the malignant behavior of cells via the Akt/mTOR pathway in hepatocellular carcinoma.

MAP17, a small non-glycosylated membrane protein, was significantly up-regulated in hepatocellular carcinoma (HCC) tissues in our previous genome-wide microarray analysis. In this study, quantitative real-time RT-PCR and immunohistochemistry were applied to examine MAP17 mRNA and protein expression in primary HCC and matched peritumoral tissues. The disease-free survival (DFS) and overall survival (OS) was estimated using the Kaplan-Meier analysis. The expression of MAP17 was significantly higher in HCC tissues compared to the paired peritumoral tissues at both mRNA and protein levels. High MAP17 expression was positively correlated with gender, distant metastasis, early recurrence (≤ 2 year), and serum alpha-fetoprotein (all p < 0.05). Kaplan-Meier analysis showed that the DFS (p = 0.004) and OS (p = 0.013) in HCC patients with elevated expression of MAP17 were much worse than that in the low expression subgroup. High level of MAP17 was also significantly associated with a high probability of HCC early recurrence after surgical resection (p = 0.005). Cox regression analysis indicated MAP17 was an independent prognostic factor for DFS (HR, 1.710; 95% CI, 1.156-2.449, p = 0.012) and OS (HR, 1.743; 95% CI, 1.152-2.639, p = 0.009) in HCC. Silencing MAP17 significantly inhibited the proliferation, invasion and migration of HCC cells in vitro, and decreased the expression levels of Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2448) and MMP-9. Suggesting MAP17 was a novel diagnostic and prognostic biomarker for HCC patients and promoted HCC cell proliferation, invasion and migration via the Akt/mTOR pathway.

Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1).

Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness, and new treatment approaches are needed. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematological malignancies and in breast tumors. Therefore, we analyzed the expression of this oncogene in sarcomas and its relationship with clinico-pathological features, as well as tested whether it can be used as a new biomarker to predict the therapeutic response to bortezomib and new therapies for sarcomas. We found that the levels of MAP17 were related to clinical features and poor survival in a cohort of 69 patients with different sarcoma types, not being restricted to any special subtype of tumor. MAP17 expression is associated with poor overall survival (p<0.001) and worse disease-free survival (p=0.002). Cell lines with high levels of MAP17 show a better response to bortezomib in vitro. Furthermore, patient-derived xenografts (PDX) with high levels of MAP17 respond to bortezomib in vivo. Our results showed that this response is due to the lower levels of NFκB and autophagy activation. Therefore, we suggest that MAP17 is a new biomarker to predict the efficacy of bortezomib as a new therapy for sarcomas.

The Possible Involvement of Epstein -Barr virus In the Etiology of Leukemia

Background: Although considerable brainstorm has been known into Epstein-Barr virus EBV as an important etiologic factor in various tumors, virtually little is known about the relationship between EBV genes and leukemia. The actual cause of Leukemia, which is a serious cancer in Sudan, is still under scrutiny. Controversial hypotheses were proposed suggesting the role of physical as well as chemical and even biological factors as being responsible for Leukemia incidents. We hypothesized that EBV could be involved in the etiology of leukemia. We describe here the results of our attempt to find a possible link between leukemia and EBV. Methods: Real-time Polymerase Chain Reaction assay (q-PCR) has recently been used widely for detection of Cell-free EBVDNA in the plasma of patients with leukemia. To determine the possible correlation between plasma cell-free EBV DNA levels and the leukemia, we studied the plasma EBV DNA levels in patients with leukemia that were presented at Radiation and Isotope Centre Khartoum (RICK), Sudan during therapy. The concentrations of plasma cell-free EBV DNA of 128 leukemic patients, 17 patients with EBV-associated lymphoid malignancies during the course of therapy Burkitt's lymphoma, Hodgkin's disease, Nasopharyngeal carcinoma (NPC) as control positive and 15 healthy controls were determined by using the (q-PCR) assay with the PrimerDesign™ genesig quantification kit. Results: The results revealed that EBV DNA was detectable in a wide range of leukemia patients. Plasma EBV DNA was detected in 33/88 Acute lymphoblastic leukemia (ALL) patients 28/40 Chronic myelogenous leukemia (CML),15/17 patients with EBV-associated lymphoid malignancies, but not in any of 15 healthy control subjects. The median concentration of EBV DNA in leukemia and healthy control groups was 6561.00 and 0.00 copies/ml, respectively. Conclusion: Our findings provided evidence of the involvement of EBV in patients with leukemia. The results suggested that EBV DNA genome encoding the non-glycosylated membrane protein BNRF1 pl43 was observed in a significant proportion of patients with ALL. However, we could not exclude a correlation between these viral infections and later leukemogenesis in childhood ALL in Sudan.

MAP17 (PDZKIP1) Expression Determines Sensitivity to the Proteasomal Inhibitor Bortezomib by Preventing Cytoprotective Autophagy and NFκB Activation in Breast Cancer.

MAP17 is a small nonglycosylated membrane protein that is overexpressed in a high percentage of carcinomas. High levels of MAP17 enhance the tumorigenic properties of tumor cells by increasing oxidative stress, which is dependent on Na(+)-coupled cotransport. Here, we show that MAP17 is associated with proteins involved in protein degradation and that proteasome inhibition induces autophagy. To analyze whether MAP17 could also alter this process, we used the proteasome inhibitor bortezomib (Velcade, PS-341), which is approved for the treatment of multiple myeloma and mantle cell lymphoma, although it has a high rate of resistance emergence and poor efficacy in solid tumors. We provide evidence that bortezomib induces a cytoprotective effect by activating autophagy and NFκB nuclear translocation, responses that are repressed in the presence of high levels of MAP17 both in vitro and in vivo. Indeed, patients with multiple myeloma treated with bortezomib showed higher response rates and a longer time to progression associated with increased levels of MAP17 expression. The MAP17-induced sensitivity to bortezomib is dependent on the oxidative status of the cells and the activity of Na(+)-coupled transporters because treatment with antioxidants or the inhibitor furosemide restores the cytoprotective activity induced by bortezomib. Therefore, bortezomib induces a prosurvival response through cytoprotective autophagy and NFκB nuclear translocation, which is repressed by high levels of MAP17. We propose that the levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematologic malignancies and in other tissues that are not commonly responsive to the drug.

MAP17 (PDZKIP1) as a novel prognostic biomarker for laryngeal cancer.

Larynx cancer organ preservation treatments with chemo and radiotherapy have substantially improved laryngoesophageal dysfunction-free survival. However, both of them lead to a high incidence of acute and chronic toxicities and a significant number of patients relapse. To date, there is no evidence available to establish the group of patients that may benefit from preservation approaches and clinical criteria such as primary tumor extension or pretreatment tracheotomy are not validated. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The tumoral behavior induced by MAP17 is associated with reactive oxygen species production in which SGLT1 seems involved. In this study we found that the levels of MAP17 were related to clinical findings and survival in a cohort of 58 patients with larynx cancer. MAP17 expression is associated with overall survival (p<0.001) and laryngoesophageal dysfunction-free survival (p=0.002). Locoregional control in patients with high MAP17 showed better outcomes than those with low MAP17 (p=0.016). Besides, a positive correlation was observed between MAP17 expression and SGLT (p=0.022) and the combination of high levels of MAP17/SGLT also led to an increased overall survival (p=0,028). These findings suggest that MAP17, alone or in combination with SGLT1, may become a novel predictive biomarker for laryngeal carcinoma.

Human Cytomegalovirus US28 Facilitates Cell-to-Cell Viral Dissemination

Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28YFP expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAGYFP in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28YFP protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28YFP is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the ΔUS28 virus (TB40/E-FLAGYFP) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAGYFP (ΔUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.

A Murine Model of Variant Late Infantile Ceroid Lipofuscinosis Recapitulates Behavioral and Pathological Phenotypes of Human Disease

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6nclf) mouse line to validate its utility for translational research. We demonstrate that this Cln6nclf mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6nclf mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics.

P38α limits the contribution of MAP17 to cancer progression in breast tumors

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.

MAP17, a ROS-dependent oncogene

MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas. Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition, decreased apoptotic sensitivity, and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis. MAP17 is overexpressed in a great variety of human carcinomas, including breast tumors. Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression. Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and, especially, for malignant progression.

MAP17 overexpression is a common characteristic of carcinomas

We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified, being overexpressed in carcinomas. We found that MAP17 is overexpressed in a great variety of human carcinomas. Immunohistochemical analysis of MAP17 during cancer progression shows, at least in prostate and ovarian carcinomas, that overexpression of the protein strongly correlates with tumoral progression (P < 0.0001). Many tumor cells also express MAP17 and its expression does not correlate with expression of SCL, a neighbor gene reported to be co-expressed in some hematopoietic cell lines. SCL neither is expressed in most MAP17-positive tumors, indicating the independent transcription of MAP17, at least in carcinomas. We cloned 5' genomic region to MAP17 and described the minimal promoter necessary to produce independent activation of MAP17. Moreover, we have found that MAP17 promoter is activated by oncogenes. Taken together, our data show an independent activation of MAP17 promoter that can be driven by oncogenes and that might explain the common overexpression of MAP17 in human carcinomas.

MAP17 enhances the malignant behavior of tumor cells through ROS increase

Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

Large scale genetic screen identifies MAP17 as protein bypassing TNF‐induced growth arrest

Although activated macrophages destroy cancer cells more effectively than normal cells, the ability to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor (TNF) alpha. Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. With the aim of identifying genes with these properties we undertook a large scale genetic screen to identify genes able to bypass TNF-induced G1 arrest. We identified MAP17, a small 17 kDa nonglycosylated membrane protein that localizes to the plasma membrane and the Golgi apparatus. Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction. However, expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells. The inhibition of TNF is specific since MAP17 does not alter the response to other cytokines such as IFNgamma. As described in the Xenopus oocyte system, MAP17 increases the uptake of mannose in some cells, but this effect is not responsible for TNF bypass. We have also analyzed the expression of MAP17 mRNA in a panel of cell lines. MAP17 is expressed in 30% of cell lines of different origin. However, MAP17 mRNA expression did not correlate with TNF resistance. Our data indicates that although MAP17 expression might bypass TNF-induced growth arrest, it is not the only determinant of this response.

The Location of Asparagine-linked Glycans on West Nile Virions Controls Their Interactions with CD209 (Dendritic Cell-specific ICAM-3 Grabbing Nonintegrin)

Mammalian cell-derived West Nile virus preferentially infects cells expressing the C-type lectin CD209L (dendritic cellspecific ICAM-3 grabbing nonintegrin-related protein; liver- and lymph node-specific ICAM-3 grabbing nonintegrin) but not cells expressing CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin). In contrast, Dengue virus infection is enhanced in cells expressing either attachment factor. The West Nile virus envelope (E) protein contains a single N-linked glycosylation site at residue 154, whereas Dengue virus E contains sites at residues 153 and 67. We introduced a glycosylation site at position 67 into West Nile virus E. Reporter virus particles pseudotyped with this E protein infected cells using either CD209 or CD209L. We also introduced glycosylation sites at several novel positions. All sites allowed CD209L-mediated infection, but only a subset promoted CD209 use. As seen for other viruses, mannose-rich glycans on West Nile virus were required for its interactions with CD209. Surprisingly, however, mannose-rich glycans were not required for CD209L-mediated infection. Complex glycans, particularly N-acetylglucosamine-terminated structures, were able to mediate reporter virus particle interactions with CD209L. We propose that CD209L recognizes glycosylated flaviviruses with broad specificity, whereas CD209 is selective for flaviviruses bearing mannose-rich glycans. The location of the N-linked glycosylation sites on a virion determines the types of glycans incorporated, thus controlling viral tropism for CD209-expressing cells.

Mannose-binding lectin binds to two major outer membrane proteins, opacity protein and porin, of Neisseria meningitidis.

Human mannose-binding lectin (MBL) provides a first line of defense against microorganisms by complement activation and/or opsonization in the absence of specific Ab. This serum collectin has been shown to activate complement when bound to repeating sugar moieties on several microorganisms, including encapsulated serogroup B and C meningococci, which leads to increased bacterial killing. In the present study, we sought to identify the meningococcal cell surface components to which MBL bound and to characterize such binding. Outer membrane complex containing both lipooligosaccharide (LOS) and proteins and LOS from Neisseria meningitidis were examined for MBL binding by dot blot and ELISA. MBL bound outer membrane complex but not LOS. The binding to bacteria by whole-cell ELISA did not require calcium and was not inhibited by N-acetyl-glucosamine or mannose. With the use of SDS-PAGE, immunoblot analysis, and mAbs specific for meningococcal opacity (Opa) proteins and porin proteins, we determined that MBL bound to Opa and porin protein B (porB). The N-terminal amino acid sequences of the two MBL binding proteins confirmed Opa and PorB. Purified PorB inhibited the binding of MBL to meningococci. Escherichia coli with surface-expressed gonococcal Opa bound significantly more MBL than did the control strain. The binding of human factor H to purified PorB was markedly inhibited by MBL in a dose-dependent manner. Meningococci incubated with human serum bound MBL as detected by ELISA. We conclude that MBL binds to meningococci by a novel target recognition of two nonglycosylated outer membrane proteins, Opa and PorB.

Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose in Xenopus laevis oocytes.

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and alpha-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.

Role of calnexin in the glycan-independent quality control of proteolipid protein.

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins.

Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP(5) (previously named G(L)), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP(2b) (formerly designated G(S)), GP(3), and GP(4). Analysis of the appearance of the GP(2b), GP(3), and GP(4) proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP(2b) and GP(4) and heterotrimers of GP(2b), GP(3), and GP(4). Shortly after their release from infected cells, virions contained mainly cystine-linked GP(2b)/GP(4) heterodimers, which were subsequently converted into disulfide-bonded GP(2b)/GP(3)/GP(4) trimers through the covalent recruitment of GP(3). This process occurred faster at a higher pH but was arrested at 4 degrees C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP(2b)/GP(3)/GP(4) trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP(3) with the cystine-linked GP(2b)/GP(4) dimer in freshly released virions, nor did we observe higher-order structures of the GP(2b)/GP(4) or GP(2b)/GP(3)/GP(4) complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP(2b)/GP(3)/GP(4) heterotrimers at 4 degrees C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP(2b)/GP(4) dimer and GP(3) was also inferred from coexpression experiments showing that the presence of GP(3) increased the electrophoretic mobility of the disulfide-bonded GP(2b)/GP(4) dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.