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Control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94 kDa (Grp94).

ABSTRACT: Bile-salt-dependent lipase (BSDL; EC 3.1.1.13) is an enzyme expressed by the pancreatic acinar cell and secreted as a component of the pancreatic juice. During its route towards secretion, BSDL is associated with intracellular membranes by means of a multiprotein folding complex, which includes the glucose-regulated protein of 94 kDa (Grp94). We have postulated that the association of BSDL with membranes is required for the complete O-glycosylation of the protein, which diverts BSDL from a degradation route and consequently allows its secretion. To further characterize the role of Grp94 in BSDL secretion, we have studied the effect of a ribozyme specifically targeted to Grp94 mRNA. This ribozyme has been transfected into AR4-2J cells, and we have shown that a decrease in Grp94 expression leads to a concomitant decrease in BSDL secretion and expression. Geldanamycin (GA), which alters Grp94 functions, also affects the release of BSDL into the culture medium of AR4-2J cells. BSDL expressed in GA-treated AR4-2J cells is unstable. Furthermore, under conditions that decrease the level of BSDL secretion, no intracellular accumulation of the enzyme was observed, suggesting that BSDL that cannot associate with (or be structured by) Grp94 could be rapidly degraded. We have further shown that this degradation probably occurs via the ubiquitin-dependent pathway. Altogether, these results indicate that Grp94 has a pivotal role in BSDL folding and in the sorting of this pancreatic enzyme.

SUBMITTER: Nganga A

PROVIDER: S-EPMC1221528 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Project description:A fetoacinar pancreatic protein (FAP) associated with the ontogenesis, differentiation and oncogenic transformation of the human exocrine pancreas has been purified from pancreatic juices of patients suffering from pancreatitis or duodenal cancers invading the pancreas [Escribano and Imperial (1989) J. Biol. Chem. 264, 21865-21871]. This protein has striking similarities, i.e. M(r), amino acid composition and N-terminal sequence, to the bile-salt-dependent lipase (BSDL) of normal human pancreatic secretion. The aim of this study was to gain further insight into the nature of the two proteins. Reactivity with the mouse monoclonal antibody J28 (mAb J28), which characterizes FAP, and enzyme activity could not be dissociated during biochemical purification of BSDL. Furthermore, a polyclonal antiserum raised against purified human BSDL reacted completely with FAP in Western-blot analysis giving additional support to the idea of similar molecular structures for BSDL and FAP. However, by the same technique, mAb J28 reacted with a relatively restricted population of BSDL molecules. The classical BSDL preparation could be separated into molecules bearing the J28 epitope and those devoid of it by immunoaffinity on immobilized mAb J28. The two subpopulations had identical N-terminal sequences and some differences in their amino acid compositions. However, they had different carbohydrate compositions. J28-epitope-bearing molecules were active on BSDL substrates, although their specific activity was decreased. These results are consistent with the existence of two closely related polypeptide chains with different glycan counterparts. Therefore, if the name FAP is reserved for molecules bearing the J28 epitope, which is linked to a carbohydrate-dependent structure. FAP could represent an oncofetal-related variant of BSDL. Our result is the first demonstration of the existence of an oncofetal-type subpopulation of an otherwise normally secreted human pancreatic enzyme.

Project description:Breast milk contains bile salt-stimulated lipase (BSSL), which significantly increases the fat digestion capacity of newborns who have limited pancreatic lipase secretion in the first few months after birth. Problematically, Holder pasteurization used in non-profit milk banks to ensure the microbiological safety of donor milk for infants, particularly preterm infants (<37 weeks gestation age), destroys milk BSSL, thus limiting infant fat absorption capacity. Alternative strategies are needed to ensure the safety of donor milk while preserving BSSL activity. Three alternative pasteurization techniques-high-pressure processing (HPP, 550 MPa, 5 min), gamma cell irradiation (IR, 2.5 Mrads) and UV-C (254 nm, 0-33,000 J/L)-were compared with Holder pasteurization (low-temperature long-time, LTLT, 62.5°C, 30 min) for retention of BSSL activity in donor breast milk. As the time required for donor milk pasteurization by UV-C in published methods was not clear, donor breast milk was spiked with seven common bacterial strains and treated by UV-C for variable time periods and the minimum UV-C dosage required to achieve a 5-log10 reduction of CFU/mL was determined. Eight thousand two hundred fifty J/L of UV-C exposure was sufficient to achieve 5-log10 reduction of each of bacterial targets, including Bacillus and Paenibacillus spores. The retention of BSSL activity was highest after HPP (retaining 62% of the untreated milk BSSL activity), followed by UV-C (16,500 J/L), IR and LTLT (35, 29, and 0.3% retention, respectively). HPP was an effective alternative to pasteurize milk with improved retention of BSSL activity compared to Holder pasteurization. Future work should investigate the effect of alternative pasteurization techniques on the entire array of bioactive components in donor breast milk and how these changes affect preterm infant health outcomes. Implementation of HPP technique at milk banks could improve donor milk-fed infant fat absorption and growth.

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Control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94 kDa (Grp94).

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