Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form.
The precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal-lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the beta-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing beta-hairpin loop.
Follo
Carlo
C
Laboratory of Molecular Pathology, Dipartimento di Scienze Mediche, Università del Piemonte Orientale A. Avogadro, via Solaroli 17, 28100 Novara, Italy.
Castino
Roberta
R
Nicotra
Giuseppina
G
Trincheri
Nicol F
NF
Isidoro
Ciro
C
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002402:Cathepsin D / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010436:Pepstatins / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D010436:Pepstatins; D011994:Recombinant Proteins; D002402:Cathepsin D; C031375:pepstatin