Abstract
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.
Highlights
(Bohmer, F.-D., Kraft, R., Otto, A., Wernstedt, C., Hell- acid sequencing [2]
Recombinant mRNA derived from this cDNA yielded a fatty acid-binding proteins (FABPs)) and on the basis of amino acid sequence differs from polypeptide that behaved like the unknown immunore- heart fatty acid-binding protein (H-FABP) in only seven positions [2, 11]
In this report we show that H-FABP and A-FABP are simultaneously expressed in lactating bovine mammary gland, and on the basis of this observation we provide evidence that the published structure can be traced back to a mixture of H-FABP and contaminating A-FABP and propose that the mammary derived growth inhibitor” (MDGI) function is exerted by the well characterized H-FABP
Summary
(Bohmer, F.-D., Kraft, R., Otto, A., Wernstedt, C., Hell- acid sequencing [2]. The amino acid sequence of the 14.5-kDa man, U., Kurtz, A., Muller, T., Rohde, K., Etzold, G., Leh- MDGI revealed no homology to any of the hitherto known mann, W., Langen, P., Heldin, C.-H., and Grosse, R. (1987) J. This protein crossreacted with antibodies against H-FABP purified from bovine heart [12] but differed from the latter at positions 12, 14, 40, 43, 93, and 127 and lacked the C-terminal amino acid (see Fig. 8).
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