Chaperone Function of a Grp 94-related Protein for Folding and Transport of the Pancreatic Bile Salt-dependent Lipase

Nadine Bruneau,Dominique Lombardo

doi:10.1074/jbc.270.22.13524

Abstract

In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion. This route was inhibited by drugs that affect protein synthesis and intracellular transport. In the presence of monensin, BSDL was solely detected in microsome membrane fractions. The association of BSDL with intracellular membranes involved a protein complex, formed by at least two proteins of 94 and 56 kDa. In cells experiencing the metabolic stress due to azetidine-2-carboxylic acid, BSDL was additionally associated with a protein of 46 kDa. Affinity blotting showed that BSDL bound directly to the 94-kDa protein (p94). It was suggested that p94 could be a molecular chaperone, further identified as related to the 94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL (i.e. BSDL bound to the Grp 94-related p94) was O- and N-glycosylated and consequently appeared released from membranes in the trans-Golgi compartment. Therefore and for the first time, it is suggested that a multiprotein complex including the chaperone Grp 94-related p94 protein may play an essential role in the folding and transport of BSDL. One hypothesis is that the association of BSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein.

Highlights

In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion
One hypothesis is that the association ofBSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein
These two agents inhibited the secretion of BSDL by 80-90%; it appeared that the secretion of the glycosylated BSDL was sensitive to energy depletion or to sodium/potassium equilibration in cells, as is the transport of a-amylase

Summary

Abbreviations used

BSDL, bile salt-dependent lipase (EC 3.1.1.-); Grp, glucose-regulated protein; Hsp, heat shock protein; Endo H, endoglycosidase H (EC 3.2.1.96). Our experiments showed that BSDL was retained without degradation within the cell upon tunicamycin or brefeldin A treatment of cells [10]. All these data suggest that the secretion or the folding of BSDL could be under the control of chaperones. Glycosylated BSDL, formed a complex with proteins of 94 kDa (to which the enzyme is bound), 56 kDa, and 46 kDa. The 94-kDa protein is probably related to the 94-kDa glucoseregulated protein or Grp

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION