Cloning and expression of rat pancreatic β-cell malonyl-CoA decarboxylase

Abstract

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

AMPK as a metabolic switch in rat muscle, liver and adipose tissue after exercise

“…Malonyl CoA decarboxylase catalyses the decarboxylation of malonyl CoA to form acetyl CoA. In liver, the enzyme is to a considerable extent localized to mitochondria and peroxisomes by specific targeting sequences (Voilley et al 1999); however, a modest but significant percentage of enzyme protein appears to be cytosolic (M. Bendayan, M. Prentki, A.K. Saha, N. Ruderman, unpublished data).…”

Malonyl‐CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes

“…A specific antibody against rat MCD [7] was raised by immunizing rabbits with a chemically derived peptide H-M-D-E-L-L-R-R-A-V-P-P-T-P-D-Y (in single acid code) corresponding to the amino acid 39-53 of the rat MCD (GenBank accession number: AJ007704) as described by Harlow and Lane [16]. IgG fractions were made from rabbit sera using Protein-A sepharose (Amershaw BioSciences, Baie dÕUrfé, QC, Canada) as described in the manufacturerÕs protocol.…”

“…MCD activity was assayed by coupling the MCD reaction to the citrate synthase and the glutamate-oxaloacetate transaminase reactions in the presence of [ 14 C]aspartate to form [ 14 C]citrate [18]. [ 14 C]citrate was separated from unreacted [ 14 C]aspartate using Dowex resin and centrifugation [7,11].…”

“…Cloning of the rat malonyl-CoA decarboxylase (MCD) revealed that the enzyme contains both mitochondrial and perox-isomal targeting signals together with a candidate translation initiation site after the mitochondrial presequence [7]. Recent studies have focused on the possibility that MCD regulates intracellular malonyl-CoA levels.…”

A Rare Case of Malonic Aciduria Diagnosed by Newborn Screening in Qatar

“…[5] and thus decrease the β-oxidation of fatty acids in both mitochondria and peroxisomes. The MLYCD gene has a tissue-specific expression pattern [6,7] with the highest message levels in the heart muscle, then the skeletal muscle, followed by the brain, small intestine, liver, kidney and pancreas. The characteristic phenotype is variable, but may include developmental delays in early childhood, seizures, hypotonia, diarrhea, vomiting, metabolic acidosis, hypoglycemia, ketosis, abnormal urinary compounds, lactic acidemia, and hypertrophic cardiomyopathy [8].…”