Apical Sorting of Bovine Enteropeptidase Does Not Involve Detergent-resistant Association with Sphingolipid-Cholesterol Rafts*
- From the Division of Hematology and Oncology, Department of Medicine and Barnes-Jewish Hospital, Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
Abstract
Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK50053.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Ave., Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax: 314-454-3012; E-mail: esadler{at}im.wustl.edu.
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↵2 D. Lu, X. Zheng, and J. E. Sadler, manuscript in preparation.
- Abbreviations:
- MDCK
-
Madin-Darby canine kidney
- BEK
-
recombinant bovine proenteropeptidase
- dSA-BEK
-
mutant proenteropeptidase lacking the amino-terminal signal anchor domain
- dSAdL-BEK
-
mutant proenteropeptidase lacking the signal anchor, mucin-like, and light chain domains
- HL-BEK
-
mutant proenteropeptidase lacking the amino-terminal signal anchor and mucin-like domains
- L-BEK
-
mutant proenteropeptidase containing the light chain and 17 carboxyl-terminal residues of the heavy chain
- NHS-SS-biotin
-
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate
- PAGE
-
polyacrylamide gel electrophoresis
- DMEM
-
Dulbecco’s modified Eagle’s medium
- MOPS
-
4-morpholinepropanesulfonic acid
- PMSF
-
phenylmethylsulfonyl fluoride
- PBS
-
phosphate-buffered saline
- BHK
-
baby hamster kidney
- PNGase
-
peptide N-glycosidase
- endo H
-
endoglycosidase H
- MES
-
4-morpholineethanesulfonic acid
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- BSA
-
bovine serum albumin.
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- Received July 17, 1998.
- Revision received September 17, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
