Immunodetection and molecular cloning of a bile-salt-dependent lipase isoform in HepG2 cells.
In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile(327) and Glu(350) of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1. 7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated.
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INSERM U 260, Unité de Recherches de Physiopathologie des Régulations Hormono-Nutritionnelles, Faculté de Médecine, 27 Bld Jean Moulin, 13385 Marseille Cedex 05, France. Alain.Verine@medecine.univ-mrs.fr
Bruneau
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Valette
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Le Petit-Thevenin
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Pasqualini
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Lombardo
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology; D015153:Blotting, Western; D003001:Cloning, Molecular; D003429:Cross Reactions; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008113:Liver Neoplasms / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001647:Bile Acids and Salts; D007527:Isoenzymes; D012333:RNA, Messenger; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase