Jrg Kuipers

Localization and intron usage analysis of the human CPT1B gene for muscle type carnitine palmitoyltransferase I

We isolated and sequenced cDNA and genomic DNA fragments of the human CPT1B gene, encoding muscle type camitine palmitoyltransferase I. A recombinant P1 phage containing CPT1B was mapped to chromosome 22qter by fluorescent in situ hybridization. This finding supports the concept that liver type and muscle type isoforms of CPT I are encoded by different loci at separate chromosomal positions. Analysis of CPT1B cDNA sequences revealed the presence of an untranslated 5 exon and differential processing of introns 13 and 19. The alternative splicing of intron 13 causes an in-frame deletion leading to a 10 amino acid residues smaller protein. Using different splice acceptor sites, intron 19 is spliced in the majority of cases, but 4 out of 14 sequenced CPT1B 3 cDNA clones contain part of intron 19 in stead of exon 20. We found that differential polyadenylation is the mechanism behind the existence of these alternative 3 CPT1B mRNA forms.

Rationale for a Conditional Knockout Mouse Model to Study Carnitine Palmitoyltransferase I Deficiencies

Several severe congenital cardiomyopathies are known to be associated with deficiencies in long-chain fatty acid transport and oxidation. Our studies are focused on a key enzyme in the regulation of intracellular long-chain fatty acid transport: carnitine palmitoyltransferase 1. Of this enzyme, two isoforms are expressed in the neonatal heart: L-CPT1 (the liver-type isoform) and M-CPT1 (the muscle-type isoform). It is known from studies in rats that chemical inhibition of both CPT1 isoforms results in hypertrophy of the cardiomyocytes, leading to an increase in heart-weight of up to 25%. With the aid of expressed sequence tag database analyses, cDNA- and genomic sequence information, we analysed the human gene for M-CPT1 in detail, and obtained partial clones of the murine genes for both CPT1 isoforms. We now started the development of a conditional knockout model to analyse and dissect deficiencies in these genes. While of the other mitochondrial components of the carnitine system deficiencies are known, some with severe cardiac consequences, M-CPT1 deficiencies have never been described. This suggests that M-CPT1 deficiency either (1) has not been recognised within the pool of congenital disorders, (2) is detrimental in an early stage of reproduction or embryogenesis, or (3) does not lead to physiological problems, probably due to the existence of a rescue system. If (1) is the case, the phenotypic effects of M-CPT1 deficiency have to be studied in order to generate criteria for clinical decision making and diagnosis. Option (2) demonstrates the necessity to use novel vector systems to create conditional gene disruptions. Hypothesis (3) implies a possible role for L-CPT1, and a knockout model allows a study of the interaction between the genes for L-CPT1 and M-CPT1. Applicable strategies to develop such a model system will be discussed.

Confocal Laser Scanning Microscopy of Human Skin Fibroblasts Showing Transient Expression of a Green Fluorescent Carnitine Palmitoyltransferase 1 Fusion Protein

The mitochondrial outer membrane enzyme carnitine palmitoyltransferase 1 (CPT1) is a main site of regulation of intracellular long-chain fatty acid transport. At least two isoforms of CPT1 are expressed in the body: L-CPT1 (the “liver-type” isoform) and M-CPT1 (the “muscle-type” isoform). Skin fibroblasts from healthy humans are known to contain only one isoform of CPT1: the liver-type, which is encoded by the gene CPT1A. Skin fibroblasts from patients with a liver-type CPT1 deficiency do not express either of the two known CPT1 isoforms (neither livernor muscle-type), and therefore could provide an excellent background to study CPT1 by means of molecular complementation. In this chapter, we describe the first experiments we carried out with a gene fusion of a complementary DNA of the human gene for muscle-type carnitine palmitoyltransferase (CPT1B) and a gene encoding an “enhanced” green fluorescent protein (GFP). We wished to express the human CPT1B gene in human skin fibroblasts, taking the following facts into consideration: