O-GLYCOSYLATION OF C-TERMINAL TANDEM-REPEATED SEQUENCES REGULATES THESECRETION 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 en zyme to which is associated the O-glycosylation, We have postulated th at the O-glycosylation of BSDL may contribute to mask PEST sequences a nd to trigger the secretion of this enzyme instead of its delivery int o a degradative pathway (Bruneau, N., and Lombardo, D. (1995) J. Biol. Chem. 270, 13524-13525). 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 K 1 wild-type line and the O-glycosylation defective CHO IdlD line. In t hese latter cells, O-glycosylation can be reversibly modulated by cult ure conditions. Results indicate that the rate of BSDL synthesis by tr ansfected CHO K1 or CHO IdlD cells reflects, independently of culture conditions, the amount of mRNA specific for BSDL present in these tran sfected cells. Nevertheless, the rate of secretion of the enzyme depen ds upon cell culture conditions and increases with the cell capability to O-glycosylate C-terminal tandem-repeated sequences, Immunoprecipit ation experiments performed on cell lysates suggested that a rapid deg radation of BSDL occurred particularly when transfected CHO IdlD cells were cultured under non-permissive conditions. We further showed that BSDL secreted by CHO IdlD cells grown under nonpermissive conditions that normally prevent O-glycosylation incorporated galactose and was r eactive with peanut agglutinin, which recognizes the core structure of O-linked glycans, We concluded that the BSDL expressed by CHO IdlD ce lls grown under non-permissive conditions was rapidly degraded but a f raction of the enzyme was allowed to O-glycosylate and consequently wa s secreted.