Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation
| Autor: | Eva Smolenova, Norbert Werth, Konrad Sandhoff, Andrej Hasilik, Hans-Wilhelm Grosch, Madanan Madathiparambil Gopalakrishnan, Silvia Locatelli-Hoops, Michael Nettersheim |
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| Rok vydání: | 2004 |
| Předmět: |
Spodoptera
Cathepsin D Biochemistry Oligomer Saposins law.invention chemistry.chemical_compound Glycolipid Multienzyme Complexes law Cell Line Tumor Animals Humans Binding site Molecular Biology Cathepsin Prosaposin Enzyme Precursors biology Chemistry U937 Cells Cell Biology biology.organism_classification Recombinant Proteins Enzyme Activation Culture Media Conditioned Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Biotinylation Recombinant DNA Dimerization Pseudogenes Research Article |
| Zdroj: | Biochemical Journal. 383:507-515 |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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 KD1 of 5.8±2.9×10−8 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. |
| Databáze: | OpenAIRE |
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