Human pancreatic cancer is characterized by an alteration in fucose-containing surface blood group antigens such as H antigen, Lewis b, Lewis y, and sialyl-Lewis. These carbohydrate determinants can be synthesized by sequential action of alpha(2,3) sialyltransferases or alpha(1,2) fucosyltransferases (Fuc-T) and alpha(1,3/1,4) fucosyltransferases on (poly)N-acetyllactosamine chains. Therefore, the expression and the function of seven fucosyltransferases were investigated in normal and cancer pancreatic tissues and in four pancreatic carcinoma cell lines. Transcripts of FUT1, FUT2, FUT3, FUT4, FUT5, and FUT7 were detected by RT-PCR in carcinoma cell lines as well as in normal and tumoral tissues. Interestingly, the FUT6 message was only detected in tumoral tissues. Analysis of the acceptor substrate specificity for fucosyltransferases indicated that alpha(1,2) Fuc-T, alpha(1,3) Fuc-T, and alpha(1,4) Fuc-T were expressed in microsome preparations of all tissues as demonstrated by fucose incorporation into phenyl beta-d-galactoside, 2'-fucosyllactose, N-acetyllactosamine, 3'-sialyl-N-acetyllactosamine, and lacto-N-biose. However, these fucosyltransferase activities varied between tissues. A substantial decrease of alpha(1,2) Fuc-T activity was observed in tumoral tissues and cell lines compared to normal tissues. Conversely, the activity of alpha(1,4) Fuc-T, which generates Lewis a and sialyl-Lewis a structures, and that of alpha(1,3) Fuc-T, able to generate a lactodifucotetraose structure, were very important in SOJ-6 and BxPC-3 cell lines. These increases correlated with an enhanced expression of Lewis a, sialyl-Lewis a, and Lewis y on the cell surface. The activity of alpha(1,3) Fuc-T, which participates in the synthesis of the sialyl-Lewis x structure, was not significantly modified in cell lines compared to normal tissues. However, the sialyl-Lewis x antigen was expressed preferentially on the surface of SOJ-6 and BxPC-3 cell lines but was not detected on Panc-1 and MiaPaca-2 cell lines suggesting that several alpha(1,3) Fuc-T might be involved in sialyl-Lewis x synthesis.

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N', N'-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.

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Bile salt-dependent lipase (BSDL, E.C. 3.1.1.-) is a digestive enzyme secreted by the pancreatic acinar cell. Once in the duodenum, the enzyme, upon activation by primary bile salts, hydrolyzes dietary lipid esters such as cholesteryl esters and lipid-soluble vitamin esters. This enzyme is partially transferred from the duodenum or pancreas to the circulation where it has been postulated to exert a systemic action on atheroma-generating oxidized-low density lipoprotein (LDL). In the present study, sera from 40 healthy normolipidemic volunteers were used to investigate the possible linkage between circulating BSDL, lipids, and lipoproteins. We showed, firstly, that pancreatic-like BSDL activity can be detected in these serums. Secondly, BSDL activity increased significantly with the level of LDL-cholesterol and was also positively linked to the serum concentration of Apo B100 and Apo A-I. Thirdly, we also established that BSDL was associated with LDL, in part by a specific interaction with Apo B100, while no interaction was found with Apo A-I. No linkage with other recorded parameters (triglycerides, phospholipids, and high density lipoprotein-cholesterol) was detected. Because an increase in LDL-cholesterol represents an important risk factor for atheroma, the concomitant increase in BSDL, which can metabolize atherogenic LDL, suggests for the first time that this circulating enzyme may exert a positive effect against atherosclerosis.

Bile-salt-dependent lipase (BSDL, EC 3.1.1.-) is an enzyme expressed by the pancreatic acinar cells and secreted as a component of the pancreatic juice of all examined species. During its secretion route BSDL is associated with intracellular membranes. This association allows the complete glycosylation of the enzyme or participates in the inhibition of the enzyme activity, which can deleterious for the acinar pancreatic cell. Thereafter, the human BSDL is phosphorylated by a serine/threonine protein kinase and released from intracellular membranes. In the present study, we show that the rat pancreatic BSDL, expressed by AR4-2J cells used as a model, is phosphorylated by a protein kinase that is insensitive to inhibitors of protein kinases A, C or G and that the phosphorylation process is favoured by okadaic acid (an inhibitor of protein phosphatases 1 and 2A). However, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), which is a specific inhibitor of casein kinase II, abolishes the phosphorylation in vitro of BSDL within micro- somes of AR4-2J pancreatic cells. We showed further that the alpha-subunit of casein kinase II co-locates with BSDL within the lumenal compartment of the Golgi. Genistein, which perturbs the trans-Golgi network, also inhibits the phosphorylation of BSDL, suggesting that this post-translational modification of BSDL probably occurred within this cell compartment. The inhibition of the phosphorylation of BSDL by DRB also decreases the rate at which the enzyme is secreted. Under the same conditions, the rate of alpha-amylase secretion was not modified. These data strongly suggest that phosphorylation is a post-translational event, which appears to be essential for the secretion of BSDL.