title: High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells.
abstract: We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal protease implicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant protein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precursor (proCDrec) that reacted with a polyclonal antibody against residues 7-21 of the propeptide sequence. In contrast to the control, in cells infected with the recombinant virus proCDrec was largely secreted into the culture medium, although it contained high-mannose oligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCDrec was processed into the 48 kDa intermediate single-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method based on Pepstatin A-affinity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on its latency in activity assay at acid pH and on its reactivity with antibodies specific for the N-terminus, the purified protein was judged to be in the inactive precursor form. During incubation at acid pH the purified proCDrec underwent autocatalytic processing and acquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCDrec specifically reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus-directed proCDrec displays structural and functional features resembling those of native human proCD. This system can therefore be exploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interesting protein.
authors.affiliation: Dipartimento di Scienze Mediche, Università A. Avogadro, via Solaroli 17, 28100 Novara, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Démoz
authors.forename: Roberta
authors.initials: R
authors.lastname: Castino
authors.forename: Carlo
authors.initials: C
authors.lastname: Follo
authors.forename: Andrej
authors.initials: A
authors.lastname: Hasilik
authors.forename: Bonnie F
authors.initials: BF
authors.lastname: Sloane
authors.forename: Ciro
authors.initials: C
authors.lastname: Isidoro
mesh_terms: D000818:Animals; D002402:Cathepsin D / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004792:Enzyme Precursors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D007106:Immune Sera; D009844:Oligosaccharides; D011994:Recombinant Proteins; C058345:procathepsin D; D002402:Cathepsin D