tailieunhanh - Báo cáo khoa học: Human acid sphingomyelinase Assignment of the disulfide bond pattern

Human acid sphingomyelinase (haSMase, EC ) catalyzes the lysosomal degradation ofsphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads toNiemann–Pick disease, a severe sphingo-lipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a fewstructural properties of haSMase have been elucidated. | Eur. J. Biochem. 270 1076-1088 2003 FEBS 2003 doi Human acid sphingomyelinase Assignment of the disulfide bond pattern Stephanie Lansmann1. Christina G. Schuette2 Oliver Bartelsen1 Joera Hoernschemever1. Thomas Linke1 Judith Weisgerber1 and Konrad Sandhoff1 1 Kekule-Institut fur Organische Chemie und Biochemie Universitat Bonn Germany 2Max-Planck-Institut fur Biophysikalische Chemie Gottingen Germany Human acid sphingomyelinase haSMase EC catalyzes the lysosomal degradation of Shhinoomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann-Pick disease a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then only a few structural properties of haSMase have been elucidated. For understanding of ts complex functions including its role in certain signaling and apoptosis events complete structural information about the enzyme is necessary. Here the identification ofthe disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI-MS analysis of the sesutiing pepiides sevealed the four disulfide bonds Cys120-Cys131 Cys385-Cys431 Cys584-Cys588 and Cys594-Cys607. Two additional disulfide bonds Cys221-Cys226 and Cys227-Cys250 which were not directly accessible by tryptic cleavage were identified by a combination ofa method ofpartial reduction and MALDI-PSD analysis. In the sphingolipid activator protein SAP -homologous N-terminal domain ofhaSMase one disulfide bond was assigned as Cys120-Cys131. The existence oftwo additional disulfide bridges in this region was proved as was expected for the known disulfide bond pattern ofSAP-type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP-type activator domain as predicted by sequence comparison Ponting . 1994 Protein Sci. 3 .