O-Glycosylation of C-terminal tandem-repeated sequences regulates the secretion of rat pancreatic bile salt-dependent lipase.
Amino acid sequences rich in Pro, Glu, Ser, and Thr (PEST) are common to rapidly degraded proteins (Rogers, S., Wells, R. & Rechsteiner, M. (1986) Science 234, 364-368). On pancreatic bile salt-dependent lipase (BSDL), PEST sequences are present in the C-terminal region of the enzyme to which is associated the O-glycosylation. We have postulated that the O-glycosylation of BSDL may contribute to mask PEST sequences and to trigger the secretion of this enzyme instead of its delivery into a degradative pathway (Bruneau, N., and Lombardo, D. (1995) J. Biol. Chem. 270, 13524-13523). To further examine the role of the O-linked glycosylation on BSDL metabolism, rat pancreatic BSDL cDNA was stably transfected into two Chinese hamster ovary (CHO) cell lines, the CHO K1 wild-type line and the O-glycosylation defective CHO ldlD line. In these latter cells, O-glycosylation can be reversibly modulated by culture conditions. Results indicate that the rate of BSDL synthesis by transfected CHO K1 or CHO ldlD cells reflects, independently of culture conditions, the amount of mRNA specific for BSDL present in these transfected cells. Nevertheless, the rate of secretion of the enzyme depends upon cell culture conditions and increases with the cell capability to O-glycosylate C-terminal tandem-repeated sequences. Immunoprecipitation experiments performed on cell lysates suggested that a rapid degradation of BSDL occurred particularly when transfected CHO ldlD cells were cultured under non-permissive conditions. We further showed that BSDL secreted by CHO ldlD cells grown under non-permissive conditions that normally prevent O-glycosylation incorporated galactose and was reactive with peanut agglutinin, which recognizes the core structure of O-linked glycans. We concluded that the BSDL expressed by CHO ldlD cells grown under non-permissive conditions was rapidly degraded but a fraction of the enzyme was allowed to O-glycosylate and consequently was secreted.
Bruneau
N
N
INSERM U260, Unité de Recherche de Physiopathologie des Régulations Hormono-Nutritionnelles, 13385 Marseille, France.
Nganga
A
A
Fisher
E A
EA
Lombardo
D
D
D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D015342:DNA Probes; D005690:Galactose / Q000378:metabolism; D018698:Glutamic Acid; D006031:Glycosylation; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011392:Proline; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D002787:Sterol Esterase; D013912:Threonine; D014162:Transfection
D003517:Cyclopentanes; D015342:DNA Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020126:Brefeldin A; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; D008985:Monensin; D011392:Proline; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase; D005690:Galactose