Pasqualini E, Caillol N, Panicot L, Mas E, Lloubes R, Lombardo D
INSERM U260, Faculté de Médecine-Timone, 27 Blv Jean Moulin, 13385 Marseille, France.
J Biol Chem. 1998 Oct 23;273(43):28208-18. doi: 10.1074/jbc.273.43.28208.
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.
在BxPC - 3和SOJ - 6胰腺肿瘤细胞系中免疫检测到了胆汁盐依赖性脂肪酶（BSDL）的特定转录本，BSDL是一种由人胰腺分泌的100 kDa糖蛋白。通过mRNA逆转录和扩增获得的片段测序证实了这些癌细胞中存在BSDL转录本。在胰腺肿瘤细胞的裂解物中检测到了该蛋白，它主要与细胞膜相关。在培养基中仅检测到极少量的该酶。免疫荧光研究表明，在SOJ - 6细胞中，BSDL与p58高尔基体蛋白共定位，提示该蛋白可能被隔离在高尔基体区室中。这些结果表明，BSDL在人胰腺肿瘤细胞中表达且无法分泌（或至少分泌能力很差）。随后，通过逆转录 - 聚合酶链反应获得了覆盖BSDL全序列的cDNA。该cDNA序列表明，由外显子1 - 10编码的N端结构域与正常人胰腺表达的BSDL相同。然而，对应于外显子11的序列（该序列应编码BSDL的16个串联重复的相同粘蛋白样序列）缺失了330个碱基对（bp），仅编码其中6个重复序列。我们得出结论，这种BSDL的截短变体将是其癌胚形式，称为胎儿胰腺腺泡蛋白。然后我们研究了330 bp的缺失是否影响该蛋白的分泌。为此，将在SOJ - 6细胞中表达的BSDL变体成熟形式的cDNA克隆到表达/分泌载体中，并转染到CHO - K1细胞中。结果表明，从SOJ - 6细胞中分离出的BSDL变体在转染细胞中表达并分泌。然而，这些转染的CHO - K1细胞分泌的BSDL水平明显高于在SOJ - 6细胞中观察到的水平。因此，在人胰腺肿瘤细胞中观察到的BSDL癌胚变体的滞留可能不是该蛋白的固有特性导致的。
