Abstract
The aspartic proteinases form a gene family whose sequences and three-dimensional structures are strongly conserved. These monomeric proteases are initially translated as prepro- precursors into the lumen of the endoplasmic reticulum (ER), whence they are post-translationally modified and sorted. We have studied three members of the gene family which are expressed in man. Procathepsin D carries N-linked oligosaccharides that are modified to contain mannose 6-phosphate residues. Mannose 6-phosphate receptors in the trans Golgi and on the plasma membrane bind and deliver the proenzyme to an acidic prelysosomal compartment and then recycle. Procathepsin D is processed to the mature enzyme and active at low pH (~4.5). Pepsinogen is the abundantly secreted precursor to gastric pepsin and is generally not glycosylated. Propeptide cleavage and activity occur extracellularly at acid pH (>3). Unlike cathepsin D and pepsin, renin shows exquisite specificity towards its physiologi-cal substrate, angiotensinogen, and is active at neutral pH in the circulation. Its glycosylation is variable, and cleavage of the propeptide takes place near neutrality at a paired basic residue site. These three aspartic proteinases share certain common features. Procathepsin D and pepsinogen are processed and active at acid pH. Procathepsin D and prorenin usually carry N-linked glycosylation sites. Procathepsin D is targeted to the lysosome, while pepsinogen and prorenin are sorted into secretory vesicles. Sorting into regulated secretory granules occurs in cells which express this specialized pathway.
We were interested in the practicality of expressing chimeras between the human proenzyme forms of cathepsin D, pepsin and renin. The studies were aimed at testing whether such chimeras would be successfully synthesized by transfected mammalian cells and whether they would be sorted through the ER-Golgi pathway and either secreted or targeted to lysosomes. Kornfeld and colleagues1 have pioneered the use of pepsinogen as a framework for the substitution of segments from procathepsin D.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Baranski, T.J., Faust, P.L., and Kornfeld, S. (1990). Generation of a lysosomal targeting signal in the secretory protein pepsinogen. Cell 63: 281–291.
Tang, J., and Wong, R.N.S. (1987). Evolution in the structure and function of aspartic proteases. J. Cell Biochem. 33: 53–63.
Davies, D.R. (1990). The structure and function of the aspartic proteinases. Annu. Rev. Biophys. Biophys. Chem. 19: 189–215.
James, M.N.G., and Sielecki, A.R. (1986). Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 Å resolution. Nature 319: 33–38.
Hartsuck, J.A., Koelsch, G., and Remington, S.J. (1992). The high-resolution crystal structure of porcine pepsinogen. Proteins: Struct. Funct. Genet. 13: 1–25.
Baker, D., Shiau, A.K., and Agard, D.A. (1993). The role of pro regions in protein folding. Curr Opin. Cell Biol. 5: 966–970.
Mercure, C, Thibault, G., Lussier-Cacan, S., Davignon, J., Schiffrin, E.L., and Reudelhuber, T.L. (1995). Molecular analysis of human prorenin prosegment variants in vitro and in vivo. J. Biol. Chem. 270:16355–16359.
Fortenberry, S.C., and Chirgwin, J.M. (1995). The propeptide is nonessential for the expression of cathepsin D. J. Biol. Chem. 270: 9778–9782.
Brechler, V., Chu, W.N., Baxter, J.O., Thibault, G., and Reudelhuber, TL. (1996). A protease processing site is essential for prorenin sorting to the regulated secretory pathway. J. Biol. Chem. 271: 20636–20640.
Klionsky, D., Banta, L., and Emr, S. (1988). Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Molec. Cell. Biol. 8: 2105–2116.
Cooper, A.A., and Stevens, T.H. (1996). VpslOp cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J. Cell Biol. 133: 529–541.
Mclntyre, G.F., Godbold, G.D., and Erickson, A.H. (1994). The pH-dependent membrane association of procathepsin L is mediated by a 9-residue sequence within the propeptide. J. Biol. Chem. 269: 567–572.
Koelsch, G., Mares, M., Metcalf, P., and Fusek, M. (1994). Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Lett. 343: 6–10.
Nishimura, Y., Takeshima, H., Sakaguchi, M., Mihara, K., Omura, T., Kato, K., and Himeno, M. (1995). Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles. J. Biochem. (Tokyo) 118: 168–177.
Dustin, M.L., Baranski, T.J., Sampath, D„ and Kornfeld, S. (1995). A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for phosphorylation of both the oligosaccharides of procathepsin D by N-acetyl glucosamine l-phosphotransferase. J. Biol. Chem. 270: 170–179.
Glickman, J.N., and Kornfeld, S. (1993). Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphocytes. J. Cell Biol. 123: 99–108.
Schorey, J.S., Fortenberry, S.C., and Chirgwin, J.M. (1995). Lysine residues in the C-terminal lobe and lysosomal targeting of procathepsin D.J. Cell Sci. 108: 2007–2015.
Barrett, A.J. (1977). Cathepsin D and other related carboxyl proteinases. In Proteinases in mammalian cells and tissues (ed. A.J. Barrett), pp. 209–248. Elsevier/North Holland Biomedical Press, Amsterdam.
Andreeva, N.S., Zdanov, A.S., Gustchina, A.E., and Fedorov, A.A. (1984). Structure of the ethanol-inhibited porcine pepsin at 2-Å resolution and binding of the methyl ester of phenylalanyl-diiodotyrsoine to the enzyme. J. Biol. Chem. 259: 11353–11365.
Šali, A., Veerapandian, B., Cooper, J.B., Moss, D.S., Hofmann, T., and Blundell, T.L. (1992). Domain flexibility in aspartic proteinases. Proteins: Struct. Funct. Genet. 12: 158–170.
Sachdev, D., Schorey, J., and Chirgwin, J. (1991). Efficient mutagenesis, expression, and purification of procathepsin D. Advan. Exp. Med. Biol. 306: 335–338.
Isidoro, C, Horst, M., Baccino, F., and Hasilik, A. (1991). Differential segregation of human and hamster cathepsin D in transfected baby-hamster kidney cells. Biochem. J. 273: 363–367.
Lin, X., Koelsch, G., Loy, A.J., and Tang, J. (1995). Rearranging the domains of pepsinogen. Protein Sci. 4: 159–166.
Westphal, V., Marcuson, E.G., Winther, J.R., Emr, S.D., and van den Hazel, H.B. (1996). Multiple pathways for vacuolar sorting of yeast proteinase A. J. Biol. Chem. 271: 11865–11870.
Marciniszyn, J., Jr., Huang, J.S., Hartsuck, J.A., and Tang, J. (1976). Mechanism of intramolecular activetion of pepsinogen. J. Biol. Chem. 251: 7095–7102.
Conner, G.E. (1992). The role of the cathepsin D propeptide in sorting to the lysosome. J. Biol. Chem. 267: 21738–21745.
Richo, G.R., and Conner, G.E. (1994). Structural requirements of procathepsin D activation and maturation. J. Biol. Chem. 269: 14806–14812.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer Science+Business Media New York
About this chapter
Cite this chapter
Chirgwin, J.M., Schultz, S., Sachdev, D. (1998). Expression of Chimeric Human Aspartic Proteinases. In: James, M.N.G. (eds) Aspartic Proteinases. Advances in Experimental Medicine and Biology, vol 436. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-5373-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-4615-5373-1_19
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-7452-7
Online ISBN: 978-1-4615-5373-1
eBook Packages: Springer Book Archive