Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation.
Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.
Gopalakrishnan
Madanan Madathiparambil
MM
Institute of Physiological Chemistry, Philipps-University Marburg, Karl-von-Frisch-Strasse 1, 35033 Marburg, Germany.
Grosch
Hans-Wilhelm
HW
Locatelli-Hoops
Silvia
S
Werth
Norbert
N
Smolenová
Eva
E
Nettersheim
Michael
M
Sandhoff
Konrad
K
Hasilik
Andrej
A
D000818:Animals; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism; D011544:Pseudogenes; D011994:Recombinant Proteins / Q000378:metabolism*; D049231:Saposins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018411:Spodoptera / Q000166:cytology; D020298:U937 Cells / Q000473:pathology
D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; C483345:PSAP protein, human; D011994:Recombinant Proteins; D049231:Saposins; C058345:procathepsin D; D002402:Cathepsin D