Biomedical subjects
Publications and source records attributed to C Hohoff.
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We succeeded in cloning the gene encoding the murine epidermal-type fatty acid binding protein (E-FABP). To avoid the screening of pseudogenes, the presence of which was shown by PCR, we designed an intron-specific probe and screened a bacterial artificial chromosome library from mouse embryonic stem cells. One of the clones obtained was analysed by restriction with various enzymes and an 11-kb EcoRI fragment with the complete gene was subcloned. The gene revealed the canonical exon/intron FABP structure consisting of four exons (112, 173, 102 and 544bp, respectively) and three introns (2217, 327 and 546bp, respectively). The exon sequences were identical with the cDNA encoding mouse E-FABP (Krieg, P., Feil, S., Fürstenberger, G., Bowden, T.G., 1993. Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterisation of a cloned sequence activated during multistage carcinogenesis in mouse skin. J. Biol. Chem. 268, 17362-17369). Of the 5' region, 2470bp were sequenced and searched for transcription factor binding sites. Putative responsive elements within the promoter region were identified that may be responsible for the wide expression observed for E-FABP in mouse tissues. The 11-kb EcoRI fragment was used to localise Fabpe on chromosome 3 in the region 3A1-3 by fluorescence in-situ hybridisation.
Members of the fatty-acid-binding protein (FABP) family are thought to play an important role in fatty acid transport within the cytosol and thus to be involved in lipid metabolism. As previous data on the occurrence of distinct FABP types in total lung are contradictory, we determined the expression of FABP types in three isolated cell types of rat lung, which are characterised by active lipid metabolism. Alveolar type-II cells synthesise, store and secrete pulmonary surfactant, a phospholipid-rich surface-tension-lowering agent, whereas lung fibroblasts, localised adjacent to the alveolar type-II cells, are assumed to provide neutral lipid substrate to alveolar type-II cells around birth, and alveolar macrophages are known to degrade complex lipids. Initial screening by reverse transcriptase PCR revealed the occurrence of heart (H-), epidermal (E-) and liver FABP in rat lung, the latter being not detectable in the three cell types studied. Cells were analysed by northern and western blotting, then quantitatively by sandwich ELISA, for which recombinant rat E-FABP was prepared. E-FABP mRNA was found in all three cell types, and E-FABP was detected in the following amounts: 240.9 +/- 19.0 ng/mg cytosolic protein in alveolar type-II cells; 172.3 +/- 0.7 ng/mg protein for lung fibroblasts; and 36.9 +/- 3.5 ng/mg protein for alveolar macrophages. This indicates a basic function of E-FABP in cellular lipid metabolism. In contrast, H-FABP probably is involved in the metabolism of neutral lipids because H-FABP mRNA was found only in lung fibroblasts with a corresponding protein level of 315.5 +/- 6.9 ng/mg. Small amounts of H-FABP protein were present in alveolar type-II cells and alveolar macrophages.
Fatty acid binding proteins (FABPs) comprise a well-established family of cytoplasmic hydrophobic ligand binding proteins and are thought to be involved in lipid metabolism by binding and intracellular transport of long-chain fatty acids. However, from other studies role for FABPs in cell signalling, growth inhibition and differentiation has also been implied. In particular, the heart-type (H-FABP) is abundantly expressed in differentiated mammary gland and its relationship with a very homologous (95%) mammary derived growth inhibitor (MDGI) was disputed. Here we give a survey on the experimental evidence for the existence of such protein with growth inhibitory function. After cloning of the bovine adipocyte-type (A-)FABP cDNA from mammary gland we conclude that the reported MDGI sequence actually represents a mixture of bovine H- and A-FABP and that the MDGI function is exerted by H-FABP. We also monitored the H-FABP level during differentiation of C2C12 muscle cells from myoblasts to multiply nucleated myotubes. H-FABP expression is clearly detected after that of the transcription factor myogenin which is upregulated immediately upon onset of differentiation and after that of the typical muscle enzyme creatine kinase. This argues against an active role of H-FABP in muscle development unlike the situation in the mammary gland.
The amino acid sequence of the mammary derived growth inhibitor (MDGI) from bovine mammary gland (Böhmer, F.-D., Kraft, R., Otto, A. , Wernstedt, C., Hellman, U., Kurtz, A., Müller, T., Rohde, K., Etzold, G., Lehmann, W., Langen, P., Heldin, C.-H., and Grosse, R. (1987) J. Biol. Chem. 262, 15137-15143) revealed 95% identity to bovine heart fatty acid-binding protein (H-FABP), explaining the observed immunocross-reactivity. However, a cDNA encoding MDGI has not been found to date. Artificial MDGI cDNA was expressed in an in vitro transcription/translation assay. Analysis by isoelectric focusing of the immunoprecipitated in vitro translation products of lactating bovine mammary gland mRNA did not indicate a protein corresponding to the in vitro translation product of artificial MDGI mRNA. Moreover, two-dimensional electrophoresis of bovine mammary gland proteins confirmed the absence of a protein with the pI of the in vitro translated artificial MDGI mRNA in bovine mammary gland and instead revealed, apart from H-FABP, an unknown protein that was recognized by anti-H-FABP antibodies. From lactating bovine mammary gland the cDNA for adipocyte fatty acid-binding protein (A-FABP) was cloned. The in vitro translation of recombinant mRNA derived from this cDNA yielded a polypeptide that behaved like the unknown immunoreactive protein. Western blotting and immunofluorescence using monospecific antibodies demonstrated the coexistence of H-FABP and A-FABP in the lactating mammary gland. Taking into account that deviations of the MDGI sequence from the bovine H-FABP sequence correspond with A-FABP we attribute the structure originally reported as MDGI to a mix of these proteins.