ABSTRACT
TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.
Subject(s)
Cation Transport Proteins , Congenital Disorders of Glycosylation , Humans , Manganese/metabolism , Galactose , Antiporters/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Cation Transport Proteins/metabolism , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolismABSTRACT
TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165's function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.
ABSTRACT
The remarkable structural diversity of glycans that is exposed at the cell surface and generated along the secretory pathway is tightly regulated by several factors. The recent identification of human glycosylation diseases related to metal transporter defects opened a completely new field of investigation, referred to herein as "metalloglycobiology", on how metal changes can affect the glycosylation and hence the glycan structures that are produced. Although this field is in its infancy, this review aims to go through the different glycosylation steps/pathways that are metal dependent and that could be impacted by metal homeostasis dysregulations.
Subject(s)
Glycomics , Glycosylation , Metals , Polysaccharides , Humans , Cation Transport Proteins/metabolism , Congenital Disorders of Glycosylation/metabolism , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Glycomics/trends , Golgi Apparatus/enzymology , Golgi Apparatus/metabolism , Homeostasis , Magnesium/chemistry , Magnesium/metabolism , Metals/chemistry , Metals/metabolism , Oxidation-Reduction , Polysaccharides/chemistry , Polysaccharides/metabolism , Zinc/chemistry , Zinc/metabolismABSTRACT
Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification.
Subject(s)
Cation Transport Proteins , Manganese , Humans , Manganese/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Antiporters/metabolism , Golgi Apparatus/metabolism , HomeostasisABSTRACT
BACKGROUND: COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. METHODS: CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients. FINDINGS: CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371-0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097-0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], -0.8180 to -0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent. INTERPRETATION: Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings. FUNDING: French National Blood Service-EFS, the Association "Les Amis de Rémi" Savigneux, France, the "Fondation pour la Recherche Médicale (Medical Research Foundation)-REACTing 2020".
Subject(s)
COVID-19 , Humans , Cohort Studies , Endothelial Cells , COVID-19 Serotherapy , Immunization, Passive , Antibodies, ViralABSTRACT
Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen-HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 - paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn't observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.
Subject(s)
COVID-19 , Pandemics , Humans , COVID-19/therapy , Endothelial Cells , Interleukin-10 , Interleukin-18 , Interleukin-1beta , Interleukin-6 , Interleukin-8 , Technology , Tumor Necrosis Factor-alpha , Ultraviolet Rays , COVID-19 SerotherapyABSTRACT
Glycosylation is a ubiquitous and universal cellular process in all domains of life. In eukaryotes, many glycosylation pathways occur simultaneously onto proteins and lipids for generating a complex diversity of glycan structures. In humans, severe genetic diseases called Congenital Disorders of Glycosylation (CDG), resulting from glycosylation defects, demonstrate the functional relevance of these processes. No real cure exists so far, but oral administration of specific monosaccharides to bypass the metabolic defects has been used in few CDG, then constituting the simplest and safest treatments. Oral D-Galactose (Gal) therapy was seen as a promising tailored treatment for specific CDG and peculiarly for TMEM165-CDG patients. TMEM165 deficiency not only affects the N-glycosylation process but all the other Golgi-related glycosylation types, then contributing to the singularity of this defect. Our previous results established a link between TMEM165 deficiency and altered Golgi manganese (Mn2+) homeostasis. Besides the fascinating power of MnCl2 supplementation to rescue N-glycosylation in TMEM165-deficient cells, D-Gal supplementation has also been shown to be promising in suppressing the observed N-glycosylation defects. Its effect on the other Golgi glycosylation types, most especially O-glycosylation and glycosaminoglycan (GAG) synthesis, was however unknown. In the present study, we demonstrate the differential impact of D-Gal or MnCl2 supplementation effects on the Golgi glycosylation defects caused by TMEM165 deficiency. Whereas MnCl2 supplementation unambiguously fully rescues the N- and O-linked as well as GAG glycosylations in TMEM165-deficient cells, D-Gal supplementation only rescues the N-linked glycosylation, without any effects on the other Golgi-related glycosylation types. According to these results, we would recommend the use of MnCl2 for TMEM165-CDG therapy.
ABSTRACT
SLC10A7, encoded by the so-called SLC10A7 gene, is the seventh member of a human sodium/bile acid cotransporter family, known as the SLC10 family. Despite similarities with the other members of the SLC10 family, SLC10A7 does not exhibit any transport activity for the typical SLC10 substrates and is then considered yet as an orphan carrier. Recently, SLC10A7 mutations have been identified as responsible for a new Congenital Disorder of Glycosylation (CDG). CDG are a family of rare and inherited metabolic disorders, where glycosylation abnormalities lead to multisystemic defects. SLC10A7-CDG patients presented skeletal dysplasia with multiple large joint dislocations, short stature and amelogenesis imperfecta likely mediated by glycosaminoglycan (GAG) defects. Although it has been demonstrated that the transporter and substrate specificities of SLC10A7, if any, differ from those of the main members of the protein family, SLC10A7 seems to play a role in Ca2+ regulation and is involved in proper glycosaminoglycan biosynthesis, especially heparan-sulfate, and N-glycosylation. This paper will review our current knowledge on the known and predicted structural and functional properties of this fascinating protein, and its link with the glycosylation process.
Subject(s)
Amelogenesis Imperfecta , Congenital Disorders of Glycosylation , Osteochondrodysplasias , Symporters , Congenital Disorders of Glycosylation/genetics , Glycosaminoglycans/genetics , Glycosylation , Humans , Organic Anion Transporters, Sodium-DependentABSTRACT
About half of the eukaryotic proteins bind biometals that participate in their structure and functions in virtually all physiological processes, including glycosylation. After reviewing the biological roles and transport mechanisms of calcium, magnesium, manganese, zinc and cobalt acting as cofactors of the metalloproteins involved in sugar metabolism and/or glycosylation, the paper will outline the pathologies resulting from a dysregulation of these metals homeostasis and more particularly Congenital Disorders of Glycosylation (CDGs) caused by ion transporter defects. Highlighting of CDGs due to defects in SLC39A8 (ZIP8) and TMEM165, two proteins transporting manganese from the extracellular space to cytosol and from cytosol to the Golgi lumen, respectively, has emphasized the importance of manganese homeostasis for glycosylation. Based on our current knowledge of TMEM165 structure and functions, this review will draw a picture of known and putative mechanisms regulating manganese homeostasis in the secretory pathway.
Subject(s)
Congenital Disorders of Glycosylation/metabolism , Golgi Apparatus/metabolism , Manganese/metabolism , Animals , Antiporters/metabolism , Biological Transport , Cation Transport Proteins/metabolism , Congenital Disorders of Glycosylation/pathology , Glycosylation , Golgi Apparatus/pathology , Homeostasis , HumansABSTRACT
Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/Mn2+ transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn2+ sensitivity. Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn2+ sensitivity.
Subject(s)
Antiporters/physiology , Cation Transport Proteins/physiology , Golgi Apparatus/metabolism , Manganese/metabolism , Calcium/metabolism , Congenital Disorders of Glycosylation/metabolism , Glycosylation , HEK293 Cells , Humans , Ion TransportABSTRACT
Congenital disorders of glycosylation are severe inherited diseases in which aberrant protein glycosylation is a hallmark. Transmembrane protein 165 (TMEM165) is a novel Golgi transmembrane protein involved in type II congenital disorders of glycosylation. Although its biologic function is still a controversial issue, we have demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi Mn2+ homeostasis defect. The goal of this study was to delineate the cellular pathway by which extracellular Mn2+ rescues N-glycosylation in TMEM165 knockout (KO) cells. We first demonstrated that after extracellular exposure, Mn2+ uptake by HEK293 cells at the plasma membrane did not rely on endocytosis but was likely done by plasma membrane transporters. Second, we showed that the secretory pathway Ca2+-ATPase 1, also known to mediate the influx of cytosolic Mn2+ into the lumen of the Golgi apparatus, is not crucial for the Mn2+-induced rescue glycosylation of lysosomal-associated membrane protein 2 (LAMP2). In contrast, our results demonstrate the involvement of cyclopiazonic acid- and thapsigargin (Tg)-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+. Interestingly, overexpression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b isoform in TMEM165 KO cells partially rescues the observed LAMP2 glycosylation defect. Overall, this study indicates that the rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ involves the activity of Tg and cyclopiazonic acid-sensitive pumps, probably the SERCA pumps.-Houdou, M., Lebredonchel, E., Garat, A., Duvet, S., Legrand, D., Decool, V., Klein, A., Ouzzine, M., Gasnier, B., Potelle, S., Foulquier, F. Involvement of thapsigargin- and cyclopiazonic acid-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+.
Subject(s)
Gene Expression Regulation/drug effects , Indoles/pharmacology , Manganese/pharmacology , Membrane Proteins/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thapsigargin/pharmacology , Antiporters , Biological Transport , Calcium/metabolism , Cation Transport Proteins , Congenital Disorders of Glycosylation/drug therapy , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , Glycosylation , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , Homeostasis , Humans , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomal-Associated Membrane Protein 2/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/geneticsABSTRACT
Congenital Disorders of Glycosylation (CDG) are rare inherited diseases causing glycosylation defects responsible for severe growth and psychomotor retardations in patients. Whereas most genetic defects affect enzymes directly involved in the glycosylation process, like glycosyltransferases or sugar transporters, recent findings revealed the impact of gene mutations on proteins implicated in both Golgi vesicular trafficking and ion homeostasis. TMEM165 is one of these deficient Golgi proteins found in CDG patients whose function in the secretory pathway has been deduced from several recent studies using TMEM165 deficient mammalian cells or yeast cells deficient in Gtd1p, the yeast TMEM165 ortholog. These studies actually confirm previous observations based on both sequence and predicted topology of this transmembrane protein and the phenotypes of human and yeast cells, namely that TMEM165 is very probably a transporter involved in ion homeostasis. Whereas the exact function of TMEM165 remains to be fully characterized, several studies hypothesize that TMEM165 could be a Golgi localized Ca2+/H+ antiporter. However, recent data also support the role of TMEM165 in Golgi Mn2+ homeostasis then arguing for a putative role of Mn2+ transporter for TMEM165 essential to achieve the correct N-glycosylation process of proteins in the secretory pathway. This manuscript is a review of the current state of knowledge on TMEM165 deficiencies in Congenital Disorders of Glycosylation as well as new data on function of TMEM165 and some speculative models on TMEM165/Golgi functions are discussed.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Golgi Apparatus/genetics , Homeostasis/genetics , Membrane Proteins/genetics , Antiporters , Cation Transport Proteins , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Glycosylation , Golgi Apparatus/pathology , Humans , Ions/metabolism , Membrane Proteins/deficiency , Protein Transport/genetics , Secretory Pathway/geneticsABSTRACT
BACKGROUND: Blood products use has increased in France between 2000 and 2011. To understand the reasons for this increase, data about transfused patients and transfusion practices needed to be updated. STUDY DESIGN AND METHODS: A nationwide cross-sectional survey was performed with health care establishments. Diagnoses and indication for the transfusion, pretransfusion laboratory results, and blood products used were collected during a randomly selected 24-hour period in 2011. All patients who received at least one blood product delivered on the survey day were included. RESULTS: A total of 10,794 blood products were requested for 4720 patients: 8688 red blood cell (RBC) units, 842 platelet (PLT) concentrates, and 1264 fresh-frozen plasma (FFP) units. Hematologic and cancer pathologies included 46% of transfused patients, 34% of the patients had transfusions in a surgical context, and 32.4% of transfused patients were receiving medication with an impact on transfusion. Nearly half of RBC transfusions were performed with hemoglobin levels of less than 8 g/dL. PLT transfusions for prophylactic indication were prescribed with PLT counts of less than 20 × 109 and 50 × 109 /L in 56.9 and 86.6% of patients, respectively. RBCs and PLTs transfusion practices were in agreement with national guidelines. FFP units were involved in 8.0% of all prescriptions. Among these, 57.4% were requested in the context of an acute hemorrhage and 8.4% for plasma exchange. The median of FFP use (n = 2) in a nonsurgical context, excluding plasma exchange, suggests an insufficient dosing of FFP. CONCLUSION: Except for insufficient FFP dosing per patient and limitations on assessment of indications for prescribing, transfusion practices were in agreement with national guidelines.
Subject(s)
Blood Transfusion/statistics & numerical data , Cross-Sectional Studies , Erythrocyte Transfusion/statistics & numerical data , France/epidemiology , Humans , Plasma , Plasma Exchange/statistics & numerical data , Platelet Transfusion/statistics & numerical data , Practice Guidelines as Topic , Surveys and QuestionnairesABSTRACT
BACKGROUND: The prevention of presyncopal and syncopal reactions to whole blood donation is important for both the donor's safety and their retention as blood donors. The best strategy to achieve this remains debated. STUDY DESIGN AND METHODS: A prospective cluster-randomized trial comparing three hydration modes (500 mL of an isotonic drink, 500 mL of water, just before phlebotomy, or advice to drink [control arm]) coupled or not with light muscle tensing exercises, was carried out in mobile and fixed units of two regional blood centers in southeast France between January and July 2014. The main outcome was the cumulative incidence of presyncope (feeling faint) and syncope (fainting) at the donation site or in the 48 hours after leaving the site. Secondary outcomes were the cumulative incidence of these adverse events during donation, immediately after blood donation, or within 48 hours. RESULTS: Overall, presyncope or syncope occurred in 5.5% of the 4576 donors. Compared to controls, drinking 500 mL (isotonic solution or water) significantly reduced the rate of events (odds ratio [OR], 0.74; 95% confidence interval [CI], 0.55-0.99; p = 0.041) independently of muscle tensing exercise. Muscle tensing exercises significantly reduced syncopal-type reactions during the donation (OR, 0.64; 95% CI, 0.42-0.98; p = 0.041), and an isotonic drink significantly reduced delayed off-site syncopal-type reactions (OR, 0.62; 95% CI, 0.40-0.98; p = 0.040) and tiredness after donation (OR, 0.75; 95% CI, 0.59-0.94; p = 0.014). CONCLUSIONS: Drinking 500mL of water or isotonic drink close to phlebotomy is useful in preventing presyncopal or syncopal reactions in blood donors. Isotonic drinks have the advantage of preventing delayed reactions and tiredness after whole blood donation.
Subject(s)
Blood Donors , Drinking/physiology , Exercise/physiology , Syncope/prevention & control , Adult , Blood Banks , Fatigue/prevention & control , Female , Humans , Incidence , Isotonic Solutions/therapeutic use , Male , Middle Aged , Muscle Tonus/physiology , Prospective Studies , Syncope/etiology , Young AdultABSTRACT
Lactoferrin is thought to be the most polyvalent protein present in host defense against tissue injuries and infections in vertebrates. Owing to the propensity of its basic N-terminal domain to interact with various microbial and host targets, lactoferrin not only has antimicrobial properties, but also modulates the innate and adaptive immune responses. Lactoferrin may indeed up- and downregulate immune cell activation, migration, and growth. Whereas the immunomodulatory properties of lactoferrin are evidenced from in vivo studies using either lactoferrin-knockout, lactoferrin-overexpressing transgenic models, and dietary lactoferrin, few mechanisms from in vitro studies have been proposed to explain these properties. The best characterized lactoferrin targets are negatively charged molecules. They encompass pro-inflammatory microbial molecules, such as pathogen-associated molecular patterns (eg, lipopolysaccharide), but also host components such as DNA, the glycosaminoglycan chains of proteoglycans, and surface cell receptors. Signaling through these receptors is thought to be the main lever used by lactoferrin to influence immune cells and cytokine-balance-controlling cell activity. This article aims to review our current understanding, though incomplete, of the many ways lactoferrin influences the complex immune machinery and the known and putative mechanisms that may explain its properties.
Subject(s)
Adaptive Immunity , Immunity, Innate , Lactoferrin/immunology , Cytokines/immunology , Cytokines/metabolism , Humans , Inflammation/immunology , Inflammation/metabolism , Lactoferrin/metabolism , Signal Transduction/immunologyABSTRACT
UNLABELLED: Most cases of hepatitis E viral (HEV) infection in developed countries are autochthonous. Nevertheless, the reported seroprevalence of HEV varies greatly depending on the geographical area and the performance of the immunoassay used. We used validated assays to determine the prevalence of anti-HEV immunoglobulin G (IgG) and IgM among 10,569 French blood donors living in mainland France and three overseas areas. Epidemiological information was collected using a specific questionnaire. We found an overall IgG seroprevalence of 22.4% (8%-86.4%) depending on the geographical area (P < 0.001). The presence of anti-HEV IgG was associated with increasing age (P < 0.001) and eating pork meat (P = 0.03), pork liver sausages (P < 0.001), game meat (P < 0.01), offal (P < 0.001), and oysters (P = 0.02). Conversely, drinking bottled water was associated with a lower rate of anti-HEV IgG (P = 0.02). Overall IgM seroprevalence was 1% (0%-4.6%). The frequency of anti-HEV IgM was higher in donors living in a high anti-HEV IgG seroprevalence area (1.9% versus 0.7%, P < 0.001) and in those eating pork liver sausage (1.4% versus 0.7%, P < 0.01), pâté (1% versus 0.4, P = 0.04), and wild boar (1.3% versus 0.7%, P < 0.01). CONCLUSION: HEV is endemic in France and hyperendemic in some areas; eating habits alone cannot totally explain the exposure to HEV, and contaminated water could contribute to the epidemiology of HEV infection in France.
Subject(s)
Blood Donors , Disease Transmission, Infectious/statistics & numerical data , Hepatitis Antibodies/blood , Hepatitis E/epidemiology , Immunoglobulin G/immunology , Adult , Chi-Square Distribution , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay , Female , France/epidemiology , Hepatitis E/etiology , Hepatitis E virus/isolation & purification , Humans , Immunoglobulin G/blood , Male , Middle Aged , Prevalence , Risk Assessment , Seroepidemiologic Studies , Statistics, Nonparametric , Surveys and QuestionnairesABSTRACT
The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 ± 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to â¼40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.
Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Nanocapsules/chemistry , Plasmids/metabolism , Polyethyleneimine/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , HEK293 Cells , Humans , Microscopy, Confocal , Paclitaxel/chemistry , Paclitaxel/toxicity , Particle Size , TransfectionABSTRACT
TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1âµ. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Point Mutation , Antiporters , Cation Transport Proteins , Cell Membrane/genetics , Cell Membrane/metabolism , Endosomes/genetics , Endosomes/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Humans , Lysosomes/genetics , Lysosomes/metabolism , Membrane Proteins/chemistry , Protein Sorting Signals , Protein TransportABSTRACT
Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca(2+) exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca(2+) homeostasis via an uncharacterized Ca(2+) transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca(2+), and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca(2+) ion transport. Furthermore, defects in TMEM165 affected both Ca(2+) and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca(2+)/H(+) antiporters and that modification of the Golgi Ca(2+) and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.
Subject(s)
Antiporters/metabolism , Calcium/metabolism , Gene Expression Regulation/physiology , Golgi Apparatus/metabolism , Homeostasis/physiology , Membrane Proteins/metabolism , Saccharomycetales/metabolism , Blotting, Western , Cation Transport Proteins , Cell Fractionation , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation/genetics , HeLa Cells , Humans , Hydrogen/metabolism , Hydrogen-Ion Concentration , Patch-Clamp Techniques , RNA, Small Interfering/geneticsABSTRACT
During evolution from prokaryotic to eukaryotic cells, compartmentalization of cellular functions has been achieved with a high degree of complexity. Notably, all secreted and transmembrane proteins travel through endoplasmic reticulum (ER) and Golgi apparatus, where they are synthesized, folded and subjected to covalent modifications, most particularly glycosylation. N-glycosylation begins in the ER with synthesis and transfer of glycan onto nascent protein and proceeds in Golgi apparatus where maturation occurs. This process not only requires the precise localization of glycosyltransferases, glycosidases and substrates but also an efficient, finely regulated and bidirectional vesicular trafficking among membrane-enclosed organelles. Basically, it is no surprise that alterations in membrane transport or related pathways can lead to glycosylation abnormalities. During the last few years, this has particularly been highlighted in genetic diseases called CDG (Congenital Disorders of Glycosylation). Alterations in mechanisms of vesicle formation due to COPII coat component SEC23B deficiency, or in vesicles tethering, caused by defects of the COG complex, but also impaired Golgi pH homeostasis due to ATP6V0A2 defects have been discovered in CDG patients. This mini review will summarize these fascinating discoveries.