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1.
Cell ; 187(14): 3585-3601.e22, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38821050

RESUMO

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.


Assuntos
Dolicóis , Dolicóis/metabolismo , Dolicóis/biossíntese , Humanos , Glicosilação , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/genética , Masculino , Mutação de Sentido Incorreto , Feminino
3.
J Biol Chem ; : 107875, 2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39395802

RESUMO

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

4.
Am J Hum Genet ; 109(8): 1484-1499, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35896117

RESUMO

Disorders of the autosomal dominant polycystic kidney disease (ADPKD) spectrum are characterized by the development of kidney cysts and progressive kidney function decline. PKD1 and PKD2, encoding polycystin (PC)1 and 2, are the two major genes associated with ADPKD; other genes include IFT140, GANAB, DNAJB11, and ALG9. Genetic testing remains inconclusive in ∼7% of the families. We performed whole-exome sequencing in a large multiplex genetically unresolved (GUR) family affected by ADPKD-like symptoms and identified a monoallelic frameshift variant (c.703_704delCA) in ALG5. ALG5 encodes an endoplasmic-reticulum-resident enzyme required for addition of glucose molecules to the assembling N-glycan precursors. To identify additional families, we screened a cohort of 1,213 families with ADPKD-like and/or autosomal-dominant tubulointerstitial kidney diseases (ADTKD), GUR (n = 137) or naive to genetic testing (n = 1,076), by targeted massively parallel sequencing, and we accessed Genomics England 100,000 Genomes Project data. Four additional families with pathogenic variants in ALG5 were identified. Clinical presentation was consistent in the 23 affected members, with non-enlarged cystic kidneys and few or no liver cysts; 8 subjects reached end-stage kidney disease from 62 to 91 years of age. We demonstrate that ALG5 haploinsufficiency is sufficient to alter the synthesis of the N-glycan chain in renal epithelial cells. We also show that ALG5 is required for PC1 maturation and membrane and ciliary localization and that heterozygous loss of ALG5 affects PC1 maturation. Overall, our results indicate that monoallelic variants of ALG5 lead to a disorder of the ADPKD-spectrum characterized by multiple small kidney cysts, progressive interstitial fibrosis, and kidney function decline.


Assuntos
Cistos , Rim Policístico Autossômico Dominante , Cistos/genética , Fibrose , Humanos , Rim/patologia , Mutação/genética , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Sequenciamento do Exoma
5.
Am J Hum Genet ; 109(2): 345-360, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35045343

RESUMO

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.


Assuntos
Cistos do Sistema Nervoso Central/genética , Defeitos Congênitos da Glicosilação/genética , Hamartoma/genética , Deficiência Intelectual/genética , Oligossacarídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Polimicrogiria/genética , alfa-Manosidase/genética , Adolescente , Alelos , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Linhagem Celular Tumoral , Cistos do Sistema Nervoso Central/metabolismo , Cistos do Sistema Nervoso Central/patologia , Vermis Cerebelar/metabolismo , Vermis Cerebelar/patologia , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Feminino , Feto , Glicosilação , Hamartoma/metabolismo , Hamartoma/patologia , Humanos , Hipotálamo/metabolismo , Hipotálamo/patologia , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Manose/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polimicrogiria/metabolismo , Polimicrogiria/patologia , Língua/metabolismo , Língua/patologia , alfa-Manosidase/deficiência
6.
Hum Mol Genet ; 31(15): 2571-2581, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35262690

RESUMO

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.


Assuntos
Retículo Endoplasmático , Bicamadas Lipídicas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Células HeLa , Humanos , Bicamadas Lipídicas/análise , Bicamadas Lipídicas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/análise , Proteínas Qc-SNARE/metabolismo , Ubiquitinas/metabolismo
7.
Hum Genet ; 141(7): 1287-1298, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34999954

RESUMO

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.


Assuntos
Amelogênese Imperfeita , Defeitos Congênitos da Glicosilação , Osteocondrodisplasias , Simportadores , Defeitos Congênitos da Glicosilação/genética , Glicosaminoglicanos/genética , Glicosilação , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio
8.
Hum Genet ; 141(7): 1279-1286, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35182234

RESUMO

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.


Assuntos
Proteínas de Transporte de Cátions , Síndromes de Imunodeficiência , Transtornos Linfoproliferativos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Magnésio/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
9.
Proc Natl Acad Sci U S A ; 116(20): 9865-9870, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31036665

RESUMO

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.


Assuntos
Proteínas de Transporte de Cátions/genética , Defeitos Congênitos da Glicosilação/genética , Adolescente , Criança , Defeitos Congênitos da Glicosilação/metabolismo , Análise Mutacional de DNA , Hexosiltransferases/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo
10.
Hum Mol Genet ; 27(17): 3029-3045, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878199

RESUMO

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.


Assuntos
Doenças do Desenvolvimento Ósseo/etiologia , Calcificação Fisiológica , Defeitos Congênitos da Glicosilação/complicações , Genômica , Glicômica , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Simportadores/genética , Adulto , Animais , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Células Cultivadas , Estudos de Coortes , Exoma , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Lactente , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Linhagem , Fenótipo , Transporte Proteico , Simportadores/metabolismo , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
11.
FASEB J ; 33(2): 2669-2679, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30307768

RESUMO

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+.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Manganês/farmacologia , Proteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologia , Antiporters , Transporte Biológico , Cálcio/metabolismo , Proteínas de Transporte de Cátions , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosilação , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Homeostase , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
12.
J Inherit Metab Dis ; 43(2): 357-366, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31415112

RESUMO

TMEM165 is involved in a rare genetic human disease named TMEM165-CDG (congenital disorders of glycosylation). It is Golgi localized, highly conserved through evolution and belongs to the uncharacterized protein family 0016 (UPF0016). The use of isogenic TMEM165 KO HEK cells was crucial in deciphering the function of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor of many glycosylation enzymes. Severe Golgi glycosylation defects are observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells and are rescued by exogenous manganese supplementation. Intriguingly, we demonstrate in this study that the observed Golgi glycosylation defect mainly depends on fetal bovine serum, particularly its manganese level. Our results also demonstrate that iron and/or galactose can modulate the observed glycosylation defects in TMEM165 KO HEK cells. While isogenic cultured cells are widely used to study the impact of gene defects on proteins' glycosylation patterns, these results emphasize the importance of the use of validated fetal bovine serum in glycomics studies.


Assuntos
Antiporters/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Glicosilação/efeitos dos fármacos , Manganês/metabolismo , Soroalbumina Bovina/farmacologia , Antiporters/genética , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Defeitos Congênitos da Glicosilação/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Transporte de Íons
13.
J Inherit Metab Dis ; 43(6): 1349-1359, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32700771

RESUMO

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.


Assuntos
alfa-Globulinas/metabolismo , Antiporters/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Defeitos Congênitos da Glicosilação/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Biomarcadores/sangue , Defeitos Congênitos da Glicosilação/sangue , Glicosilação , Humanos , Isoformas de Proteínas/metabolismo
14.
Biochem J ; 476(21): 3281-3293, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31652305

RESUMO

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.


Assuntos
Antiporters/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Antiporters/genética , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/genética , Citosol/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Manganês/metabolismo , Mutação , Proteólise , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
15.
Am J Hum Genet ; 98(2): 322-30, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26833330

RESUMO

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.


Assuntos
Fosfatase Alcalina/metabolismo , Colesterol/metabolismo , Complexo de Golgi/genética , Homeostase , Proteínas de Membrana/deficiência , Transaminases/metabolismo , Adulto , Sequência de Aminoácidos , Ceruloplasmina/metabolismo , Retículo Endoplasmático/metabolismo , Exoma , Fibroblastos/metabolismo , Genótipo , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Fenótipo , Adulto Jovem
16.
Am J Hum Genet ; 98(2): 310-21, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26833332

RESUMO

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.


Assuntos
Complexo de Golgi/genética , Homeostase , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Exoma , Feminino , Fibroblastos/citologia , Glicosilação , Complexo de Golgi/metabolismo , Células HeLa , Heterozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Med Genet ; 55(2): 137-142, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28848061

RESUMO

The conserved oligomeric Golgi (COG) complex consists of eight subunits organized in two lobes: lobe A (COG1-4) and lobe B (COG5-8). The different functional roles of COG lobe A and lobe B might result in distinct clinical phenotypes in patients with COG-CDG (congenital disorders of glycosylation). This hypothesis is supported by three observations. First, knock-down of COG lobe A components affects Golgi morphology more severely than knock-down of COG lobe B components. Second, nearly all of the 27 patients with lobe B COG-CDG had bi-allelic truncating mutations, as compared with only one of the six patients with lobe A COG-CDG. This represents a frequency gap which suggests that bi-allelic truncating mutations in COG lobe A genes might be non-viable. Third, in support, large-scale exome data of healthy adults (Exome Aggregation Consortium (ExAC)) underline that COG lobe A genes are less tolerant to genetic variation than COG lobe B genes. Thus, comparable molecular defects are more detrimental in lobe A COG-CDG than in lobe B COG-CDG. In a larger perspective, clinical phenotypic severity corresponded nicely with tolerance to genetic variation. Therefore, genomic epidemiology can potentially be used as a photographic negative for mutational severity.


Assuntos
Defeitos Congênitos da Glicosilação/etiologia , Complexos Multiproteicos/genética , Mutação , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Defeitos Congênitos da Glicosilação/genética , Feminino , Estudos de Associação Genética , Humanos , Masculino , Complexos Multiproteicos/química , Fenótipo
18.
Hum Mol Genet ; 25(8): 1489-500, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27008884

RESUMO

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.


Assuntos
Proteínas Fúngicas/genética , Complexo de Golgi/fisiologia , Manganês/farmacologia , Proteínas de Membrana/deficiência , Mutação , Antiporters , Proteínas de Transporte de Cátions , Defeitos Congênitos da Glicosilação/genética , Proteínas Fúngicas/metabolismo , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Manganês/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo
19.
Breast Cancer Res Treat ; 171(3): 581-591, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29971627

RESUMO

PURPOSE: Cancer cells often elicit a higher glycolytic rate than normal cells, supporting the development of glycolysis inhibitors as therapeutic agents. 2-Deoxyglucose (2-DG) is used in this context due to its ability to compete with glucose. However, many studies do not take into account that 2-DG inhibits not only glycolysis but also N-glycosylation. Since there are limited publications on 2-DG mechanism of action in breast cancer, we studied its effects in breast cancer cell lines to determine the part played by glycolysis inhibition and N-linked glycosylation interference. METHODS AND RESULTS: 2-Deoxyglucose behaved as an anticancer agent with a similar efficiency on cell number decrease between the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cells. It also interfered with the N-linked glycosylation process in both cell lines as illustrated by the migration profile of the lysosomal-associated membrane protein 2 and calumenin. These results are reinforced by the appearance of an abnormal Man7GlcNAc2 structure both on lipid-linked oligosaccharides and N-linked glycoproteins of 2-DG incubated MDA-MB-231 cells. Besides, 2-DG-induced a transient endoplasmic reticulum stress that was more sustained in MDA-MB-231 cells. Both changes were abrogated by mannose. 2-DG, even in the presence of mannose, decreased glycolysis in both cell lines. Mannose partially reversed the effects of 2-DG on cell numbers with N-linked glycosylation interference accounting for 37 and 47% of 2-DG anti-cancerous effects in MDA-MB-231 and MCF-7 cells, respectively. CONCLUSION: N-linked glycosylation interference and glycolysis disruption both contribute to the anticancer properties of 2-DG in breast cancer cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glucose/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/química , Humanos , Células MCF-7
20.
Electrophoresis ; 39(24): 3133-3141, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29947113

RESUMO

Congenital disorders of glycosylation (CDG) are heterogeneous group of genetic protein and lipid glycosylation abnormalities. With some 33 reported patients, MAN1B1-CDG belongs to the more frequent causes of CDG-II. MAN1B1 encodes an α1,2-mannosidase that removes the terminal mannose residue from the middle branch. Several methods have been proposed to characterize the glycosylation changes. In MAN1B1-CDG, the abnormal accumulating N-glycan structures are mostly absent or found in trace amounts in total human serum. To overcome this issue, in this study, we present a straightforward procedure based on the use of Endo-ß-N-acetylglucosaminidase H to easily diagnose MAN1B1-CDG patients and mannosidase defects.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Glicômica/métodos , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/análise , Sequência de Carboidratos , Humanos , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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