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Project
Zebrafish as a Model for Glycosylation Defects. Fishing for the Role of TMEM165
Congenital Disorders of Glycosylation (CDG) represent a heterogeneous group of inherited diseases characterized by defective N-and O-glycosylation and defective lipid glycosylation. Protein N-glycosylation disorders, which range in severity and systemic presentation, are traditionally divided into two groups. The largest group, type I CDG (CDG-I), results from defects in enzymes involved in either the biosynthesis of the lipid-linked oligosaccharide precursor (LLO) Glc3Man9GlcNAc2-P-P-dolichol (G3M9Gn2-P-P-Dol) or its transfer to the nascent polypeptides in the ER. Thesecond group, type II CDG (CDG-II), results from alterations in the processing or remodeling of the protein-bound glycan chains. Type II CDG encompasses defects in glycosidases, glycosyltransferases, and sugar nucleotide transporters, but also in proteins involved in Golgi trafficking, structure, and pH homeostasis. In light of the ability of the latter group to alter the global environment necessary for proper glycosylation, such defects typically result in structural changes within multiple classes of glycans (e.g. N- and O-glycans). One particular CDG, caused by mutations in TMEM165, a transmembrane protein whose function remains elusive, is the subject of this thesis.
Mutations in the protein TMEM165 (transmembrane protein 165) were recently shown to cause a novel type II CDG. Two siblings presented psychomotor retardation, dwarfism, and significant skeletal abnormalities. Isoelectric focusing of serum transferrin revealed an abnormal pattern consistent with type II CDG, prompting efforts to identify the genetic basis for the disease in these patients. Glycomic analysis of N-glycans revealed an increase in the relative abundance of undersialylated and undergalactosylated glycans. Homozygosity mapping and gene array expression profiling uncovered a homozygous, deep intronic splice mutation (c.792+182G>A) within the previously uncharacterized transmembrane protein TMEM165 in the two affected siblings. Sequencing of TMEM165 in a group of unsolved CDG-IIx patients identified three additional patients with mutations: one patient with the previously described homozygous mutation (c.792+182G>A) and two unrelated individuals with missense mutations (one homozygous for c.377G>A (p.R126H) and another compound heterozygous for c.376C>T (p.R126C) and c.911G>A (p.G304R)).
The TMEM165 gene encodes a 324 amino acid protein containing 6 transmembrane-spanning domains. It is ubiquitously expressed and highly conserved within eukaryotes. TMEM165 belongs to the UPF0016 family of integral membrane proteins of unknown function. Based on predicted topology and comparative phylogeny, it is proposed to function in Golgi proton/calcium transport. Furthermore, studies in yeast have suggested that TMEM165 is a member of Golgi-localized Ca2+/H+ antiporters, and may play an important role in the maintenance of the Golgi structure and/or pH. Despite growing insight into the molecular function of TMEM165, the physiological relevance of this protein during development, particularly with regard tothe skeletal system, remains poorly understood. In an effort to improveour understanding of its physiologic and pathogenic functions, we employed a morpholino-based approach to reduce tmem165 expression in developing zebrafish.
Our results indicate that this protein is essential forproper cartilage development, as tmem165-deficient embryos exhibit altered morphology of multiple craniofacial structures. In situ hybridization demonstrated that loss of Tmem165 was associated with reduced expression of the chondroitin sulfate proteoglycan aggrecan, as well as several later stage markers of both cartilage and bone maturation. These deficiencies resulted in reduced mineralization of morphant cartilages. Although the cartilage phenotypes were effectively rescued by introduction of wild type tmem165 mRNA, no recovery was observed with tmem165 mRNA bearing the R126H change found in human patients, confirming the pathogenic nature of this mutation. Mass spectrometric analyses of control and morphant embryos demonstrated that tmem165 is necessary for proper processing of N-glycans in zebrafish, a finding consistent with the CDG phenotype seen in human patients.
In conclusion, the present work reports our study of the developmental impact of tmem165 deficiency in zebrafish, which establishes the first animal model for this new CDG subtype. Our analysis of tmem165 morphant embryos demonstrates that tmem165 deficiency in zebrafish results in altered N-linked glycosylation and cartilage defects. Importantly, both the morphological and biochemical phenotypes noted in the zebrafish model mirror those seen in human patients. This also unequivocally establishes the causal link between the TMEM165 defect and the disease.
Mutations in the protein TMEM165 (transmembrane protein 165) were recently shown to cause a novel type II CDG. Two siblings presented psychomotor retardation, dwarfism, and significant skeletal abnormalities. Isoelectric focusing of serum transferrin revealed an abnormal pattern consistent with type II CDG, prompting efforts to identify the genetic basis for the disease in these patients. Glycomic analysis of N-glycans revealed an increase in the relative abundance of undersialylated and undergalactosylated glycans. Homozygosity mapping and gene array expression profiling uncovered a homozygous, deep intronic splice mutation (c.792+182G>A) within the previously uncharacterized transmembrane protein TMEM165 in the two affected siblings. Sequencing of TMEM165 in a group of unsolved CDG-IIx patients identified three additional patients with mutations: one patient with the previously described homozygous mutation (c.792+182G>A) and two unrelated individuals with missense mutations (one homozygous for c.377G>A (p.R126H) and another compound heterozygous for c.376C>T (p.R126C) and c.911G>A (p.G304R)).
The TMEM165 gene encodes a 324 amino acid protein containing 6 transmembrane-spanning domains. It is ubiquitously expressed and highly conserved within eukaryotes. TMEM165 belongs to the UPF0016 family of integral membrane proteins of unknown function. Based on predicted topology and comparative phylogeny, it is proposed to function in Golgi proton/calcium transport. Furthermore, studies in yeast have suggested that TMEM165 is a member of Golgi-localized Ca2+/H+ antiporters, and may play an important role in the maintenance of the Golgi structure and/or pH. Despite growing insight into the molecular function of TMEM165, the physiological relevance of this protein during development, particularly with regard tothe skeletal system, remains poorly understood. In an effort to improveour understanding of its physiologic and pathogenic functions, we employed a morpholino-based approach to reduce tmem165 expression in developing zebrafish.
Our results indicate that this protein is essential forproper cartilage development, as tmem165-deficient embryos exhibit altered morphology of multiple craniofacial structures. In situ hybridization demonstrated that loss of Tmem165 was associated with reduced expression of the chondroitin sulfate proteoglycan aggrecan, as well as several later stage markers of both cartilage and bone maturation. These deficiencies resulted in reduced mineralization of morphant cartilages. Although the cartilage phenotypes were effectively rescued by introduction of wild type tmem165 mRNA, no recovery was observed with tmem165 mRNA bearing the R126H change found in human patients, confirming the pathogenic nature of this mutation. Mass spectrometric analyses of control and morphant embryos demonstrated that tmem165 is necessary for proper processing of N-glycans in zebrafish, a finding consistent with the CDG phenotype seen in human patients.
In conclusion, the present work reports our study of the developmental impact of tmem165 deficiency in zebrafish, which establishes the first animal model for this new CDG subtype. Our analysis of tmem165 morphant embryos demonstrates that tmem165 deficiency in zebrafish results in altered N-linked glycosylation and cartilage defects. Importantly, both the morphological and biochemical phenotypes noted in the zebrafish model mirror those seen in human patients. This also unequivocally establishes the causal link between the TMEM165 defect and the disease.
Date:1 Oct 2009 → 25 Jun 2014
Keywords:glycosylation
Disciplines:Genetics, Systems biology, Molecular and cell biology, Medical imaging and therapy, Other paramedical sciences
Project type:PhD project