Molecular cloning of the oncofetal isoform of the human pancreatic bile salt-dependent lipase.
Specific transcripts for bile salt-dependent lipase (BSDL), a 100-kDa glycoprotein secreted by the human pancreas, were immunodetected in BxPC-3 and SOJ-6 pancreatic tumoral cell lines. Sequencing of fragments, obtained by mRNA reverse transcription and amplification, confirmed the presence of BSDL transcripts in these cancer cells. The protein was detected in lysates of pancreatic tumoral cells, where it was mainly associated with membranes. Only a minute amount of the enzyme was detected in the culture media. Immunofluorescence studies demonstrated that in SOJ-6 cells, BSDL colocates with the p58 Golgi protein and suggested that the protein may be sequestrated within the Golgi compartment. These results demonstrated that BSDL is expressed in human pancreatic tumoral cells and cannot be secreted (or for the least very poorly). Subsequently, a cDNA covering the entire sequence of BSDL was obtained by reverse transcription-polymerase chain reaction. The sequence of this cDNA indicated that the N-terminal domain encoded by exons 1-10 was identical to that of BSDL expressed by the human normal pancreas. However, the sequence corresponding to exon 11, which should code for the 16 tandem-repeated identical mucin-like sequences of BSDL, was deleted by 330 base pairs (bp) and encoded only 6 of these repeated sequences. We conclude that this truncated variant of BSDL would be its oncofetal form, referred to as feto-acinar pancreatic protein. We then investigated whether the deletion of 330 bp affected the secretion of the protein. For this purpose, the cDNA corresponding to the mature form of the BSDL variant expressed in SOJ-6 cells was cloned into an expression/secretion vector and transfected into CHO-K1 cells. Results indicated that the variant of BSDL isolated from SOJ-6 cells was expressed and secreted by transfected cells. However, the level of BSDL secreted by these transfected CHO-K1 cells was significantly higher than that observed for SOJ-6 cells. Consequently, the retention of the oncofetal variant of BSDL observed in human pancreatic tumoral cells might not result from inherent properties of the protein.
Pasqualini
E
E
INSERM U260, Faculté de Médecine-Timone, 27 Blv Jean Moulin, 13385 Marseille, France.
Caillol
N
N
Panicot
L
L
Mas
E
E
Lloubes
R
R
Lombardo
D
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002451:Cell Compartmentation; D006023:Glycoproteins / Q000235:genetics*; D006056:Golgi Apparatus; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008049:Lipase; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D002787:Sterol Esterase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D002352:Carrier Proteins; D006023:Glycoproteins; D007527:Isoenzymes; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; C054621:CEL protein, human; D008049:Lipase