Molecular cloning, and characterization and expression of dihydrolipoamide acetyltransferase component of murine pyruvate dehydrogenase complex in bile duct cancer cells.
BACKGROUND
The association between the dihydrolipoamide acetyltransferase component (E2) of pyruvate dehydrogenase complex (PDC) and primary biliary cirrhosis (PBC) is clinically established. However, the detailed pathological function of the PDC-E2 gene is as yet unclear. In order to study the gene function in knockout and transgenic mouse models, we cloned and characterized the mouse PDC-E2 (mPDC-E2) gene. Because the expression level of PDC-E2 was elevated in PBC bile duct cells, we tried to construct a bile duct carcinoma cell line that overexpressed PDC-E2 as a PBC cell model.


METHODS
The mPDC-E2 cDNA was obtained by the 3'Race method. We overexpressed this gene in KMBC cells, using a retrovirus vector. The transcript and translated protein of mPDC-E2 were detected by Northern blot and Western blot, respectively.


RESULTS
The deduced amino-acid sequence from the cloned cDNA indicated that the fully mature protein consisted of 557 amino-acid residues, with a calculated molecular mass of 59kD. This mature protein was highly consistent with those of previously reported rat and human PDC-E2, which possessed three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. Mouse fibroblast NIH3T3 cells expressed one species of mPDC-E2 mRNA, 3.5kb in length. We also successfully constructed a stable KMBC cell line overexpressing the PDC-E2.


CONCLUSIONS
This is the first report of the mPDC-E2 sequence and is valuable for further investigation of PDC-E2 gene function in transgenic or knockout mouse models. The PDC-E2 overexpressing KMBC cell line can be used to study alterations in signal transduction or gene expression profiles in PBC bile duct.
Department of Gastroenterology and Cancer Gene Regulation, Kanazawa University Graduate School of Medical Sciences, Japan.
Liqun
L
Wang
Shuichi
S
Kaneko
Makiko
M
Kagaya
Hideki
H
Ohno
Masao
M
Honda
Kenichi
K
Kobayashi
D000123:Acetyltransferases / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001650:Bile Duct Neoplasms / Q000235:genetics*; D003001:Cloning, Molecular; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D024101:Mitochondrial Proteins; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis* / Q000235:genetics*; D051381:Rats; D014407:Tumor Cells, Cultured
D024101:Mitochondrial Proteins; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; C494878:Dlat protein, mouse