Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity - PubMed (original) (raw)
Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity
P E Milhiet et al. Biochem J. 1994.
Abstract
Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.
Similar articles
- Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.
Chevallier S, Ahn J, Boileau G, Crine P. Chevallier S, et al. Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):731-8. doi: 10.1042/bj3170731. Biochem J. 1996. PMID: 8760356 Free PMC article. - The astacin family of metalloendopeptidases.
Bond JS, Beynon RJ. Bond JS, et al. Protein Sci. 1995 Jul;4(7):1247-61. doi: 10.1002/pro.5560040701. Protein Sci. 1995. PMID: 7670368 Free PMC article. Review. - Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.
Milhiet PE, Chevallier S, Corbeil D, Seidah NG, Crine P, Boileau G. Milhiet PE, et al. Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):683-8. doi: 10.1042/bj3090683. Biochem J. 1995. PMID: 7626036 Free PMC article. - Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.
Corbeil D, Milhiet PE, Simon V, Ingram J, Kenny AJ, Boileau G, Crine P. Corbeil D, et al. FEBS Lett. 1993 Dec 13;335(3):361-6. doi: 10.1016/0014-5793(93)80420-y. FEBS Lett. 1993. PMID: 8262184 - Polarised expression of human intestinal N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin) alpha and beta subunits in Madin-Darby canine kidney cells.
Eldering JA, Grünberg J, Hahn D, Croes HJ, Fransen JA, Sterchi EE. Eldering JA, et al. Eur J Biochem. 1997 Aug 1;247(3):920-32. doi: 10.1111/j.1432-1033.1997.00920.x. Eur J Biochem. 1997. PMID: 9288916
Cited by
- Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.
Chevallier S, Ahn J, Boileau G, Crine P. Chevallier S, et al. Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):731-8. doi: 10.1042/bj3170731. Biochem J. 1996. PMID: 8760356 Free PMC article. - Characterization of the soluble, secreted form of urinary meprin.
Beynon RJ, Oliver S, Robertson DH. Beynon RJ, et al. Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):461-5. doi: 10.1042/bj3150461. Biochem J. 1996. PMID: 8615815 Free PMC article. - The astacin family of metalloendopeptidases.
Bond JS, Beynon RJ. Bond JS, et al. Protein Sci. 1995 Jul;4(7):1247-61. doi: 10.1002/pro.5560040701. Protein Sci. 1995. PMID: 7670368 Free PMC article. Review. - The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases.
Stöcker W, Grams F, Baumann U, Reinemer P, Gomis-Rüth FX, McKay DB, Bode W. Stöcker W, et al. Protein Sci. 1995 May;4(5):823-40. doi: 10.1002/pro.5560040502. Protein Sci. 1995. PMID: 7663339 Free PMC article. Review. - Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.
Milhiet PE, Chevallier S, Corbeil D, Seidah NG, Crine P, Boileau G. Milhiet PE, et al. Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):683-8. doi: 10.1042/bj3090683. Biochem J. 1995. PMID: 7626036 Free PMC article.
References
- Cell. 1981 Jan;23(1):175-82 - PubMed
- J Biol Chem. 1991 Mar 25;266(9):5540-6 - PubMed
- J Biol Chem. 1991 Sep 15;266(26):17350-7 - PubMed
- Arch Biochem Biophys. 1991 Nov 1;290(2):549-53 - PubMed
- Biochemistry. 1991 Oct 22;30(42):10065-74 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources