Insights into TMEM165 function in Golgi glycosylation and lysosomal Mn-induced degradation

Les déficits congénitaux de la glycosylation (CDG) sont un groupe de maladies génétiques rares, résultant d’altérations dans le processus de biosynthèse des glycannes. En 2012, un nouveau sous-type de CDG de type II, le TMEM165-CDG, conséquence de mutations dans le gène codant pour la protéine transmembranaire TMEM165 est découvert. Cette protéine est localisée au niveau de l’appareil de Golgi et serait le principal transporteur de manganèse dans l’appareil de Golgi. Nous savons désormais que le défaut de glycosylation du TMEM165-CDG est due à une altération de l’homéostasie du manganèse et que ce défaut peut être restauré par une supplémentation en cet ion. De façon intéressante, de fortes concentrations en manganèse entrainent la dégradation de TMEM165 au niveau des lysosomes.Au cours de mon doctorat, nous avons identifiés les acides aminés de TMEM165 responsables de sa fonction dans la glycosylation et sa sensibilité au manganèse, essentiellement deux séquences « signature » de la famille UPF0016. Nous avons également démontré pour la première fois que TMEM165 avait un lien fonctionnel avec la protéine SPCA1, une pompe calcium/manganèse golgienne. Une absence de SPCA1 engage TMEM165 dans la voie de la dégradation lysosomale.Congenital Disorders of Glycosylation (CDG) are a group of rare genetic diseases affecting the process of glycans biosynthesis. In 2012, a new subtype of CDG type II was discovered, TMEM165-CDG, resulting from mutations in the gene encoding TMEM165, a transmembrane protein. This protein is localized in the Golgi apparatus and might be the major Golgi manganese importer. The glycosylation defect observed in TMEM165-CDG results of a lack of manganese in this compartment, that is restored by manganese supplementation. Interestingly, high manganese concentrations lead to the degradation of TMEM165 in the lysosomes.During my PhD, we have identified the amino acids of TMEM165 implied in the function in glycosylation and its manganese sensitivity, essentially the two signature motifs of the UPF0016 family. We also demonstrated the first evidence of a functional link between TMEM165 and SPCA1, a Golgi calcium/manganese pump. The absence of SPCA1 targets TMEM165 to the lysosomes for degradation

Investigating the functional link between TMEM165 and SPCA1

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Involvement of thapsigargin– and cyclopiazonic acid–sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn 2+

International audienceCongenital 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+

Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity

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Association between arterial stiffness and Loa loa microfilaremia in a rural area of the Republic of Congo: A population-based cross-sectional study (the MorLo project).

BackgroundLoa loa filariasis (loiasis) is still considered a relatively benign disease. However, recent epidemiologic data suggest increased mortality and morbidity in L. loa infected individuals. We aimed to examine whether the density of L. loa microfilariae (mfs) in the blood is associated with cardiovascular disease.MethodologyUsing a point-of-care device (pOpmètre), we conducted a cross-sectional study to assess arterial stiffness and peripheral arterial disease (PAD) in 991 individuals living in a loiasis-endemic rural area in the Republic of the Congo. Microfilaremic individuals were matched for age, sex and village of residence with 2 amicrofilaremic subjects. We analyzed markers of arterial stiffness (Pulse-Wave Velocity, PWV), PAD (Ankle-Brachial Index, ABI) and cardiovascular health (Pulse Pressure, PP). The analysis considered parasitological results (L. loa microfilarial density [MFD], soil-transmitted helminths infection, asymptomatic malaria and onchocerciasis), sociodemographic characteristics and known cardiovascular risk factors (body mass index, smoking status, creatininemia, blood pressure).Principal findingsAmong the individuals included in the analysis, 192/982 (19.5%) and 137/976 (14.0%) had a PWV or an ABI considered out of range, respectively. Out of range PWV was associated with younger age, high mean arterial pressure and high L. loa MFD. Compared to amicrofilaremic subjects, those with more than 10,000 mfs/mL were 2.17 times more likely to have an out of range PWV (p = 0.00). Factors significantly associated with PAD were older age, low pulse rate, low body mass index, smoking, and L. loa microfilaremia. Factors significantly associated with an elevation of PP were older age, female sex, high average blood pressure, low pulse rate and L. loa microfilaremia.ConclusionA potential link between high L. loa microfilaremia and cardiovascular health deterioration is suggested. Further studies are required to confirm and explore this association

Flowchart of the study.

BackgroundLoa loa filariasis (loiasis) is still considered a relatively benign disease. However, recent epidemiologic data suggest increased mortality and morbidity in L. loa infected individuals. We aimed to examine whether the density of L. loa microfilariae (mfs) in the blood is associated with cardiovascular disease.MethodologyUsing a point-of-care device (pOpmètre), we conducted a cross-sectional study to assess arterial stiffness and peripheral arterial disease (PAD) in 991 individuals living in a loiasis-endemic rural area in the Republic of the Congo. Microfilaremic individuals were matched for age, sex and village of residence with 2 amicrofilaremic subjects.We analyzed markers of arterial stiffness (Pulse-Wave Velocity, PWV), PAD (Ankle-Brachial Index, ABI) and cardiovascular health (Pulse Pressure, PP). The analysis considered parasitological results (L. loa microfilarial density [MFD], soil-transmitted helminths infection, asymptomatic malaria and onchocerciasis), sociodemographic characteristics and known cardiovascular risk factors (body mass index, smoking status, creatininemia, blood pressure).Principal findingsAmong the individuals included in the analysis, 192/982 (19.5%) and 137/976 (14.0%) had a PWV or an ABI considered out of range, respectively. Out of range PWV was associated with younger age, high mean arterial pressure and high L. loa MFD. Compared to amicrofilaremic subjects, those with more than 10,000 mfs/mL were 2.17 times more likely to have an out of range PWV (p = 0.00). Factors significantly associated with PAD were older age, low pulse rate, low body mass index, smoking, and L. loa microfilaremia. Factors significantly associated with an elevation of PP were older age, female sex, high average blood pressure, low pulse rate and L. loa microfilaremia.ConclusionA potential link between high L. loa microfilaremia and cardiovascular health deterioration is suggested. Further studies are required to confirm and explore this association.</div

Results from logistic regression model explaining the presence of Peripheral Arterial Disease.

Results from logistic regression model explaining the presence of Peripheral Arterial Disease.</p

Distribution of the main characteristics according to Pulse Wave Velocity status (using References values #2).

Abbreviations: PWV, pulse wave velocity; N, number; SD, Standard deviation; MFD, microfilarial density; IQR, interquartile range; N/A, not applicable. * Chi-2 for categorical variable and Kruskal-Wallis rank test for continuous variables. ** An individual is defined as out of range if his/her PWV is higher than the 90th percentile of the population considered healthy in the same age category (see S1 Table 1 –References values #2). (DOCX)</p

Analysis of correlations between <i>Loa loa</i> microfilarial status and densities, lipid profile and glycated hemoglobin.

1 Fisher’s exact test. 2 Cramér’s V. 3 Cuzick’s test. * For lipid panel, an individual is considered out of range if at least one of the lipids is out of range (see S2 and S3 Tables). (DOCX)</p