Characterization of TRAPPC11 and GOSR2 mutations in human fibroblasts

Eukaryotic cells consist of membrane-bounded organelles which communicate with each other through vesicles for the movement of proteins and lipids between them in a process called membrane traffic. It requires different types of proteins to facilitate docking and fusion of the cargo-containing vesicles to the correct compartment. Among them are a diverse group of membrane proteins called tethering factors including multi-subunit tethering complexes (MTCs). The transport protein particle (TRAPP) complexes are a family of related MTCs that are conserved from yeast to humans. Subunits of the mammalian TRAPP complexes are known to be involved in ER-to-Golgi and intra-Golgi trafficking while playing a fundamental role in Golgi morphology. The TRAPPC11 subunit of the mammalian TRAPP III complex has been implicated in ER-to-ERGIC trafficking as well as autophagy. The mammalian TRAPP subunits are linked to a broad range of diseases of which the mechanism and the cause are not fully understood. In this study, homozygous and compound heterozygous mutations in the TRAPPC11 gene in human fibroblasts from five individuals were characterized using several biochemical, immunofluorescence and live cell microscopy techniques to identify the defective pathways and the effect of the mutations at the cellular level. Included in this study were two individuals with GOSR2 mutations displaying similar clinical features to patients with TRAPPC11 mutations. We hypothesized that the TRAPPC11 mutations would result in a number of different defects at the cellular level given the number of pathways TRAPPC11 has been suggested to function within. The current study suggests that some of the TRAPPC11 mutations are linked to a variety of cellular phenotypes including hypoglycosylation of proteins, ER-to-Golgi trafficking defects, delay in the exit of proteins from the Golgi, Golgi fragmentation, defects in the autophagy pathway as well as partial disassembly of the complex. Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex 2 (GOSR2) is a protein located in the cis-Golgi to facilitate docking and fusion of COPII vesicles from the ER. The current study also suggests that some of the GOSR2 mutations are linked to hypoglycosylation of proteins, ER-to-Golgi trafficking defects as well as a delay in the exit of proteins from the Golgi. The affected individuals showed novel mutations in TRAPPC11 and GOSR2 as well as mutations seen in previous studies. Some of the earlier TRAPPC11 mutations were in or near the foie gras domain, and these new TRAPPC11 mutations cluster near that region. This suggests that the foie gras domain plays a critical role in the function of the TRAPPC11 protein. Since, TRAPPC11 mutations affect the brain, eyes, liver, muscle, and bone, this suggests that the TRAPPC11 protein has a function in multiple tissue types and organs as well as homeostasis of the organism. This study and previous studies of TRAPPC11 lead to the conclusion that TRAPPC11 mutations, in general, result in neuromuscular phenotypes. In conclusion, these mutations can be added to the growing group of mutations in TRAPPC11 and GOSR2 causing neuromuscular and myopathy phenotypes. A better understanding of these mutations which result in neuromuscular phenotypes will allow for the screening of individuals who carry these mutations and further investigate the mechanism and treatment for these diseases

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy

Abstract Background Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Case presentation Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. Conclusion Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy

Abstract Background Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Case presentation Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. Conclusion Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy