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Originally published In Press as doi:10.1074/jbc.M003521200 on June 2, 2000

J. Biol. Chem., Vol. 275, Issue 33, 25577-25584, August 18, 2000
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N-Linked Oligosaccharides on the Meprin A Metalloprotease Are Important for Secretion and Enzymatic Activity, but Not for Apical Targeting*

Tomoko KadowakiDagger §, Takayuki TsukubaDagger , Greg P. Bertenshaw, and Judith S. Bond

From the Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-0850

The alpha  and beta  subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; ~25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin alpha  and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin alpha . Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75-100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin alpha  (alpha -(1-445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin alpha .

* This work was supported by National Institutes of Health Grant DK 19691 (to J. S. B.) and by the American Heart Association (to G. P. B.).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.

Dagger Present address: Dept. of Pharmacology, Kyushu University Faculty of Dentistry, Fukuoka 812-8582, Japan.

§ Research Fellow of the Japan Society for the Promotion of Science.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, H171, Pennsylvania State University College of Medicine, Hershey, PA 17033-0850. Tel.: 717-531-8586; Fax: 717-531-7072; E-mail: jbond@psu.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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