Isolation of novel animal cell lines defective in glycerolipid biosynthesis reveals mutations in glucose-6-phosphate isomerase.
Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 --> Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.
Haller
Jorge F
JF
Department of Physiology and Biophysics, Boston University School of Medicine, Massachusetts 02118, USA.
Smith
Conor
C
Liu
Dailan
D
Zheng
Hongying
H
Tornheim
Keith
K
Han
Gil-Soo
GS
Carman
George M
GM
Zoeller
Raphael A
RA
D000740:Anemia / Q000201:enzymology / Q000235:genetics; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism; D008661:Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics; D009468:Neuromuscular Diseases / Q000201:enzymology / Q000235:genetics; D000073718:Pancreatitis-Associated Proteins; D010743:Phospholipids / Q000096:biosynthesis* / Q000235:genetics; D017354:Point Mutation; D014280:Triglycerides / Q000096:biosynthesis* / Q000235:genetics
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; D010743:Phospholipids; C000612141:REG3A protein, human; D014280:Triglycerides; D005956:Glucose-6-Phosphate Isomerase