Site-directed mutations in the FAD-binding domain of glycerophosphate dehydrogenase: catalytic defects with preserved mitochondrial anchoring of the enzyme in transfected COS-7 cells.
Single-strand conformational polymorphism analysis of mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH) gene has revealed mutations in both the calcium- and FAD-binding domains of this enzyme in some diabetic patients. It was now investigated whether site-directed mutations in the FAD-binding domain of the mGDH gene may affect the mitochondrial anchoring and catalytic activity of the enzyme. COS-7 cells were transfected with plasmid cDNA coding for either wild-type or mutated human mGDH (G --> A substitutions at positions 352, 355, and 364 and A --> C substitution at position 390) fused, when required, at the N-terminus of green fluorescent protein. The activity of mGDH was measured by both radioisotopic ((3)HOH production from l-[2-(3)H]glycerol 3-phosphate) and colorimetric (iodoformazan formation) procedures. In cells transfected with the mGDHwt-EGFP or mGDHmut-EGFP constructs, the fused protein was found by confocal microscopy exclusively in the mitochondria, colocalized with a mitochondrial marker. In homogenates of COS-7 cells transfected with mGDHmut, however, the catalytic activity of the enzyme was decreased, this coinciding with low ratios between both the activities measured in the absence/presence of exogenous FAD and the results obtained by the colorimetric/radioisotopic procedure. Thus, although the present site-directed mutations of the mGDH gene failed to impair the mitochondrial anchoring of the enzyme, they led to catalytic defects that were, in some respect, comparable to those previously encountered in the lymphocytes or islets of type 2 diabetic patients.
Gudayol
Monica
M
Endocrinology and Diabetes Unit, IDIBAPS, Hospital Clinic de Barcelona, Barcelona, Spain.
Fabregat
Marta E
ME
Rasschaert
Joanne
J
Sener
Abdullah
A
Malaisse
Willy J
WJ
Gomis
Ramon
R
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002384:Catalysis; D006224:Cricetinae; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D005182:Flavin-Adenine Dinucleotide; D005993:Glycerolphosphate Dehydrogenase