Cell Surface Membrane Proteins Research Articles

SEC23-SEC31 the Interface Plays Critical Role for Export of Procollagen from the Endoplasmic Reticulum

COPII proteins are essential for exporting most cargo molecules from the endoplasmic reticulum. The membrane-facing surface of the COPII proteins (especially SEC23-SEC24) interacts directly or indirectly with the cargo molecules destined for exit. As we characterized the SEC23A mutations at the SEC31 binding site identified from patients with cranio-lenticulo-sutural dysplasia, we discovered that the SEC23-SEC31 interface can also influence cargo selection. Remarkably, M702V SEC23A does not compromise COPII assembly, vesicle size, and packaging of cargo molecules into COPII vesicles that we have tested but induces accumulation of procollagen in the endoplasmic reticulum when expressed in normal fibroblasts. We observed that M702V SEC23A activates SAR1B GTPase more than wild-type SEC23A when SEC13-SEC31 is present, indicating that M702V SEC23A causes premature dissociation of COPII from the membrane. Our results indicate that a longer stay of COPII proteins on the membrane is required to cargo procollagen than other molecules and suggest that the SEC23-SEC31 interface plays a critical role in capturing various cargo molecules.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Characterization of Immunodominant Membrane Protein Gene from Peanut Witches’ Broom Phytoplasma and Bait Plasmid Construction

The molecular mechanisms involving insect-vector-phytoplasma-host-plant relationships were poorly understood.Our study focused on a cell-surface membrane protein of the phytoplasma named immunodominant membrane protein(Imp).The objective of experiment was to get the basic information between the mechanism of phytoplasma-host-plant relationships.The target ORF had been amplified using primers designed based on the conserved regions of imp gene from phytoplasma.The amplified gene was analyzed for phylogenetic tree,and the encoding protein was analyzed for transmembrane region,hydrophilic and hydrophobic region in this study.The imp gene was inserted into pBT vector including λcI gene.Construction of the pBT-Imp plasmid can be used for experiment followed by conformed that imp-λcI fusing protein was expressed by IPTG induction,western blot and self-validation testing.The immunodominant membrane protein gene from peanut witches' broom(PnWB) phytoplasma was 519 bp in length,encoding a predicted protein of 172 amino acids.Homology analysis of sequence from PnWB and 17 phytoplasma showed that imp gene of PnWB phytoplasma closely related to SPWB and WBDL phytoplasma,with homology rate of nucleotide being 99.9% and 79.8%,amino acids sequence,being 100% and 70.2%.Analysis of protein structure showed that the protein from PnWB phytoplasma possesses a region with C-terminal hydrophobic transmembrane anchor and N-terminal hydrophilic characterization,possibly internal to the phytoplasma cell.

Overlapping Roles and Collective Requirement for the Coreceptors GAS1, CDO, and BOC in SHH Pathway Function

Secreted Hedgehog (HH) ligands signal through the canonical receptor Patched (PTCH1). However, recent studies implicate three additional HH-binding, cell-surface proteins, GAS1, CDO, and BOC, as putative coreceptors for HH ligands. A central question is to what degree these coreceptors function similarly and what their collective requirement in HH signal transduction is. Here we provide evidence that GAS1, CDO, and BOC play overlapping and essential roles during HH-mediated ventral neural patterning of the mammalian neural tube. Specifically, we demonstrate two important roles for these molecules: an early role in cell fate specification of multiple neural progenitors and a later role in motor neuron progenitor maintenance. Most strikingly, genetic loss-of-function experiments indicate an obligatory requirement for GAS1, CDO, and BOC in HH pathway activity in multiple tissues.

Abstract 5108: Quantitative proteomic analysis of cell-surface membrane proteins: Biomarker discovery in endometrial cancer

Abstract Background. Endometrial cancer is one of the most common malignancies of the female genital tract. The identification of proteins for prognostic assessment and therapeutic targets in this disease is of significant clinical importance. Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels and post-translational modifications that are associated with tumors. To date, proteomic studies of cancer have focused particularly on sub-cellular compartments or post-translational modifications. An analysis of the differential expression of cell-surface membrane proteins would be of particular interest, since these proteins play key roles in cell function including cell migration and drug resistance. Purpose of study. To identify membrane proteins overexpressed in endometrial cancer which may represent novel biomarkers of disease and therapeutic targets. Procedures. We developed a biotinylation-based approach for cell membrane enrichment combined with iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify cell surface proteins overexpressed in seven endometrial cancer cell lines compared with a control normal endometrial cell line. Results. 365 proteins were identified, of which, 65 proteins (18%) possessed a trans-membrane domain. We identified overexpression of 24 membrane proteins in endometrial cancer cell lines compared with a normal endometrial cell line. By this proteomic approach, we identified several tumor antigens which included antigens previously associated with endometrial cancer and antigens not previously reported in this disease. Tumorigenic proteins such as neural cell adhesion molecule L1 (NCAML1), previously reported to be overexpressed in endometrial cancer, were identified. Interestingly we also identified Claudin-4, a protein associated with poor prognosis in various cancers, but not previously reported in the pathogenesis of cancer. In addition, we also successfully identified novel antigens associated with endometrial cancer. Conclusions. We developed a high throughput cell-surface membrane proteomic analysis combining biotinylation of cell-surface membrane protein and iTRAQ technology. In addition to the identification of previously reported tumor antigens such as NCAML1 and Claudin-4, we also identified novel tumor antigens in endometrial cancer by this methodology. This iTRAQ based quantitative proteomic analysis represented a useful approach in screening for novel disease biomarkers and therapeutic targets. Further studies on the physiological role of novel tumor antigens overexpressed in endometrial cancer and their application as biomarkers/therapeutic targets are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5108. doi:10.1158/1538-7445.AM2011-5108

Vimentin/cardiolipin complex as a new antigenic target of the antiphospholipid syndrome

AbstractAntiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent abortions, and antiphospholipid antibodies (aPL). However, it is possible to find patients with clinical signs of APS who persistently test negative for aPL (seronegative APS, or SN-APS). The aim of this study was to identify new antigenic target(s) of autoantibodies in APS patients, which may also be recognized in SN-APS. We tested sera from patients with SN-APS with a proteomic approach by analyzing endothelial cell-surface membrane proteins. Sera from SN-APS patients revealed 2 reactive spots corresponding to vimentin, a protein that is shown to bind cardiolipin in vitro. Antivimentin/cardiolipin antibodies were tested in 29 SN-APS patients, 40 APS patients, 30 patients with systemic lupus erythematosus, 30 with rheumatoid arthritis, 30 with venous or arterial thrombosis, and 32 healthy control patients. We observed that not only a large proportion of SN-APS patients but also almost all the APS patients displayed the presence of antivimentin/cardiolipin antibodies. To verify the possible pathogenic role of these autoantibodies, we demonstrated that affinity-purified antivimentin/cardiolipin antibodies induced interleukin receptor-associated kinase phosphorylation and nuclear factor-κB activation in endothelial cells. Our results prompt to identify vimentin as a “new” cofactor for aPL, which may represent a useful tool mainly in SN-APS patients.

Signaling through LRP1: Protection from atherosclerosis and beyond

The low-density lipoprotein receptor-related protein (LRP1) is a multifunctional cell surface receptor that belongs to the LDL receptor (LDLR) gene family and that is widely expressed in several tissues. LRP1 consists of an 85-kDa membrane-bound carboxyl fragment (β chain) and a non-covalently attached 515-kDa (α chain) amino-terminal fragment. Through its extracellular domain, LRP1 binds at least 40 different ligands ranging from lipoprotein and protease inhibitor complex to growth factors and extracellular matrix proteins. LRP-1 has also been shown to interact with scaffolding and signaling proteins via its intracellular domain in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. LRP-1 is thus implicated in two major physiological processes: endocytosis and regulation of signaling pathways, which are both involved in diverse biological roles including lipid metabolism, cell growth/differentiation processes, degradation of proteases, and tissue invasion. The embryonic lethal phenotype obtained after target disruption of the LRP-1 gene in the mouse highlights the biological importance of this receptor and revealed a critical, but yet undefined role in development. Tissue-specific gene deletion studies also reveal an important contribution of LRP1 in vascular remodeling, foam cell biology, the central nervous system, and in the molecular mechanisms of atherosclerosis.

Purkinje Cell Death After Uptake of Anti-Yo Antibodies in Cerebellar Slice Cultures

Paraneoplastic cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic cerebellar degeneration.

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

The growing recognition of immunotherapy-responsive seizure disorders with autoantibodies to specific neuronal proteins

The concept of epilepsy and seizure disorders caused by autoantibodies to specific neuronal membrane proteins has developed significantly during the past few years. Antibodies to cell-surface membrane proteins such as voltage-gated potassium channels or N-methyl-D-aspartate receptors, or to glutamic acid decarboxylase, are found in patients with different forms of limbic encephalitis, and in a few patients with epilepsy as their main or only condition. Many of these patients do not show a good response to conventional antiepileptic drugs, but respond to immunotherapies. By contrast, studies of other antibodies in idiopathic forms of epilepsy, or epilepsy associated with systemic lupus erythematosus or coeliac disease, have not in general disclosed consistent, clinically helpful results. There are a growing number of specific antibodies associated with new onset epilepsy. These patients are likely to have an immune-mediated disorder that may benefit from immunotherapies. In autoimmune diseases such as systemic lupus erythematosus or coeliac disease, antibodies to specific membrane targets may also prove to be important in the future.

Proteomic Analysis of Human U937 Cell Line Activation Mediated byHaemophilus influenzaeType b P2 Porin and Its Surface-Exposed Loop 7

The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

Editorial: CEACAM1: fine-tuned for fine-tuning

Editorial: CEACAM1: fine-tuned for fine-tuning

Molecularly Targeted Therapies for Dysimmune Neuropathies

Conventional treatment options, including corticosteroids, intravenous immunoglobulin, or plasma exchange, often fail to treat dysimmune neuropathies, such as chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, and monoclonal gammopathy with its subtypes. Therefore, a significant percentage of patients require adjunctive immunosuppressive therapies. Considering that even immunosuppressive agents often are ineffective and/or associated with significant toxicities, the need for the development of safe and effective new treatment options is rising. Currently, several monoclonal antibodies (MAbs) have been tested in open-label small-sized studies or even in single cases so as to establish future directions in the therapy of diseases of the peripheral nervous system (PNS). Rituximab, an MAb targeting against the B cell surface membrane protein CD20, is the most widely used and promising MAb for the treatment of dysimmune neuropathies, especially for those in which immunoglobulin M (IgM) autoantibodies are pathogenetically involved. The efficacy of alemtuzumab, bevacizumab, and etanercept to treat various forms of dysimmune neuropathies is currently under investigation. This review looks critically at recent developments in molecularly targeted therapies for dysimmune neuropathies and also highlights areas of future research to pursue.

Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic cell line by SILAC followed by mass spectrometry analysis enabled identification and quantification of proteins that were differentially expressed in the two cell lines. Dual stable isotopic labels ((13)C-arginine and (13)C-lysine) instead of a single label ((13)C-arginine) increased the percentage of proteins that could be quantified from 40 to 93%. Repeated LC-MS/MS analyses (3-4 times) of each sample increased the number of identified proteins by 60%. The use of Percoll/sucrose density separation allowed subfractionation of membranes leading to enrichment of membrane proteins (66%) and reduction from 33% to only 16% of protein from other membranous organelles such as endoplasmatic reticulum, Golgi, and mitochondria. In total, our optimized methods resulted in 1919 protein identifications (corresponding to 826 at similarity level 80% (SL80); 1145 (509 at SL80) were identified by two or more peptides of which 622 (300 at SL80) were membrane proteins. The quantitative proteomic analysis identified 16 cell surface proteins as potential markers of the ability of breast cancer cells to form distant metastases.

Membrane protein quality control in post‐Golgi compartments

Elimination of non‐native polypeptides is critical for protein quality control (QC) and fundamental to preserve cellular proteostasis. The "peripheral" QC of cell surface membrane proteins (MP) remains poorly understood. To uncover the principles of MPQC in post‐Golgi compartments, we engineered chimeric CD4 molecules containing wt and temperature‐sensitive mutant l domain. The mutant was accumulated at the cell surface and unfolded at the non‐permissive temperature. Increased internalization and impaired recycling accounts for the metabolic destabilization of the unfolded chimera at the cell surface. The postendocytic sorting of the internalized chimeras was conformational‐dependent; unfolding redirected the chimera from constitutive recycling towards lysosomal degradation. Ubiquitination plays a pivotal role in the non‐native chimera degradation since; a) the chimera ubiquitination was enhanced several fold upon unfolding, 2) depletion of ubiquitin‐dependent endosomal sorting machinery (Hrs, Stam‐2 and Tsg101) delayed lysosomal delivery and 3) physical and functional association of the misfolded variant with an E3 Ub‐ligase could be identified. We propose a working model for the peripheral QC of MPs with a central role in cellular proteostasis and the etiology of conformational diseases. Supported by CIHR.

Understanding the mechanism responsible for γ6‐dependent modulation of low voltage activated Ca2+ channels by exploring physical interactions

The γ6 subunit of voltage dependent Ca2+ channels is known to decrease Ca2+ current when co‐expressed with the low voltage activated Ca2+ channel subunit α3.1. This effect of γ6 is specific to current density as there is no change in the biophysical properties of the channels when measured at the macroscopic level. Previous studies using chimeric and mutant γ subunits indicate that the first transmembrane domain (TM1) of γ6 and more specifically a GxxxA motif within it are critical for γ6's inhibitory function. In this study, cell surface membrane protein biotinylation, purification, and quantitative western blot demonstrate that the functional effect γ6 confers upon Cav3.1 current is not due to a change in the plasma membrane expression of α3.1. Furthermore, co‐immunoprecipitation assay demonstrates a physical association of γ6 and α3.1 in both HEK cells and atrial myocytes. Subsequently, we engineered chimeric and mutant subunits of γ6 to identify potential interaction site(s). Results from these experiments indicate the functionally critical domain and motif of γ6 are not required for γ6 interaction with α3.1. Further studies are required to determine the region(s) in γ6 involved in interaction with α3.1, as well as the γ6 interaction site within α3.1. Supported by a GIA from the AHA.

Targeting of G protein-coupled receptors to the plasma membrane in health and disease

G protein-coupled receptors (GPCRs) are cell surface membrane proteins that recognize specific signals (ligands) from an immense number of chemically diverse substances. These receptors act as signal transducers for messages carried by external, systemic, or local stimuli. As complex molecular structures, which must attain specific shapes, newly synthesized GPCRs are subjected to conformational scrutiny at the endoplasmic reticulum level before their passage to the plasma membrane. Such a quality control mechanism guards against aberrant protein structures and checks for proper folding, processing and structural integrity of nascent proteins. Despite this stringent quality control screening mechanism, gain- or loss-of-function mutations that result in GPCR misfolding in the endoplasmic reticulum can manifest themselves as profound effects on health. Understanding the molecular, cellular and energetic mechanisms controlling GPCR intracellular routing is essential for preventing or correcting the conformational abnormalities associated with disease-causing misfolded receptors. This article reviews the mechanisms subserving plasma membrane targeting of GPCRs and describes novel and promising approaches to correct misfolding and misrouting related to various disease states.

Functional roles of short sequence motifs in the endocytosis of membrane receptors

Internalization and trafficking of cell-surface membrane receptors and proteins into subcellular compartments is mediated by specific short-sequence signal motifs, which are usually located within the cytoplasmic domains of these receptor and protein molecules. The signals usually consist of short linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. The complex arrays of signals and recognition proteins ensure the dynamic movement, accurate trafficking, and designated distribution of transmembrane receptors and ligands into intracellular compartments, particularly to the endosomal-lysosomal system. This review summarizes the new information and concepts, integrating them with the current and established views of endocytosis, intracellular trafficking, and sorting of membrane receptors and proteins. Particular emphasis has been given to the functional roles of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into the subcellular compartments. The specific characteristics and functions of short-sequence motifs, including various tyrosine-based, dileucine-type, and other short-sequence signals in the trafficking and sorting of membrane receptors and membrane proteins are presented and discussed.

A hybrid total internal reflection fluorescence and optical tweezers microscope to study cell adhesion and membrane protein dynamics of single living cells

The dynamics of cell surface membrane proteins plays an important role in cell-cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where optical tweezers are used to trap cells and precisely control in space and time the initiation of interactions between a cell and a functionalized surface. This approach is combined with total internal reflection fluorescence microscopy to monitor dynamics of membrane bound proteins. We demonstrate an accuracy of approximately 2 s in determining the onset of the interaction. Furthermore, we developed a data analysis method to determine the dynamics of cell adhesion and the organization of membrane molecules at the contact area. We demonstrate and validate this approach by studying the dynamics of the green fluorescent protein tagged membrane protein activated leukocyte cell adhesion molecule expressed in K562 cells upon interaction with its ligand CD6 immobilized on a coated substrate. The measured cell spreading is in excellent agreement with existing theoretical models. Active redistribution of activated leukocyte cell adhesion molecule is observed from a clustered to a more homogenous distribution upon contact initiation. This redistribution follows exponential decay behaviour with a characteristic time of 35 s.

Genome sequencing and analysis of cell surface membrane proteins of phytoplasmas

Genome sequencing and analysis of cell surface membrane proteins of phytoplasmas