Rapid purification of human lysosomal membranes, characterisation of the detergent resistent microdomains, purification and reconstitution of the vacuolar proton pump (V-ATPase)

The lysosomal membrane is a dynamic environment where specific interactions among proteins and between proteins and lipids occur. These interactions are necessary for the proper functioning of the lysosomal apparatus that allows the passage of molecules into and out of the lysosomes for the degradation and recycling. From the previous studies on human placental lysosomes, occurrence of detergent resistant microdomains, in which special proteins including the acetyl-CoA: α-glucosaminide N-acetyl transferase are localised has been reported by Taute et al. (2002). In an attempt to characterise the proteins that are part of such microdomains from the lysosomal membranes, the V-ATPase was found to be enriched in fractions containing the DRMs. This protein complex catalyses an ATP dependent proton translocation and is responsible for the acidification of the lysosomal lumen. It is known that lipids and lipid peroxidation products influence the biological activity of the V-ATPase that may contribute to age-related macular degeneration, a major cause of vision loss as reported by Kopitz et al. (2004). In this work a method was established for the isolation and reconstitution of the human V-ATPase which should allow further studies on the role of special lipids and lipid derivatives on the macular degeneration. To isolate the V-ATPase, a simple and efficient system for purifying the lysosomal membrane proteins was sought as the commonly used procedures yield lysosomal preparations that are contaminated with mitochondria. To reduce the contamination, a substrate-induced selective disruption of the lysosomal vesicles followed by aggregating and sedimenting the bulk of contaminating membranes, especially those from the mitochondria, was developed. A purification of the lysosomal membrane proteins up to 300 fold as compared to the placental homogenate, based on the specific activity of the membrane-associated ß-glucosidase was achieved using this novel and convenient procedure. The lysosomal membranes thus prepared may also be used for the selective isolation and characterisation of lysosomal membrane proteins that are not yet studied in detail, like the acetyl-CoA: α-glucosaminide N-acetyl transferase and Niemann- Pick C1 protein known to be involved in the degradation of heparan sulphate and in cholesterol transport, respectively. From the purified lysosomal membranes, the V-ATPase was extracted using Triton X-100 and CHAPS and further purified by gel filtration. This is the first report on the purification of V-ATPase from a human tissue. Methyl-ß-cyclodextrin was included during the extraction step to deplete cholesterol and thereby to disrupt the microdomains. Since a number of the V-ATPase subunits possess a basic isoelectric point, and as such are difficult to be analysed by the common two-dimensional electrophoretic systems, a novel CETAB/SDS-PAGE system was used for the proteomic characterisation of the purified V-ATPase. The V-ATPase activity fractions were devoid of all the major proteins present in the lysosomal preparation used for gel filtration, though two other DRM-associated proteins, placental alkaline phosphatase and stomatin were identified pointing towards the possibility of different types of DRMs on the lysosomal or the contaminating membranes, that were not removed during the purification steps. The purified protein complex was successfully reconstituted into unilamellar vesicles. When the vesicles containing lecithin and cholesterol were used for the reconstitution, a recovery of upto 30 % was observed in the incorporated fraction, as compared to 14 % when the experiment was performed with cholesterol-free vesicles. Finally, the dose-dependant inhibitory effects of the lipofuscin componenent, A2-E were confirmed with the reconstituted V-ATPase. The ATP hydrolysis by the enzyme was completely inhibited at an A2-E concentration of 10 µM. This result extends the previous findings using cell cultures on the inhibition of V-ATPase by A2-E described by Bergmann et al. suggesting a role of A2-E in the pathogenesis of age-related macular degeneration. The inhibition of ATP hydrolysis and thereby of the lysosomal acidification may cause an accumulation of lipofuscin within the lumen of the lysosomes and thus contribute to the degeneration of the retinal epithelium. The purified and reconstituted enzyme may facilitate further studies on its inhibitory and protective agents

Untersuchung der Nutzung unterschiedlicher Transkriptionsstartpunkte für mRNA von humanem Kathepsin D unter dem Einfluß von Calcitriol

In der vorliegenden Arbeit wurden die Transkriptionsstartstellen für mRNA von humanem Kathepsin D unter dem Einfluß von Calcitriol in U937-Zellen untersucht. Zu diesem Zweck wurde die Methode der kompetitiven RT-PCR der Fragestellung entsprechend modifiziert. Die Methode der kompetitiven PCR basiert auf der kompetitiven Co-Amplifikation einer spezifischen Target-Sequenz (DNA bzw. revers transkribierte RNA) - also der zu bestimmenden Ziel-DNA-Menge - zusammen mit bekannten Mengen eines internen Standards (Kompetitor, Standard-DNA) im selben Reaktionsgefäß. Die Methode erlaubt den Nachweis kleinster Mengen von DNA bzw. RNA und zugleich deren Quantifizierung. Der dazu benötigte Kompetitor konnte mittels In-vitro-Mutagenese entwickelt werden, bis auf eine Deletion von 30 Bp ist er identisch mit der zu bestimmenden Sequenz. Es wurde mRNA aus Calcitriol-stimulierten U937-Zellen und aus Kontroll-U937-Zellen isoliert und untersucht. Mit dieser Arbeit konnte gezeigt werden, daß in beiden Fällen mehrere Transkriptionsstartstellen für Kathepsin D benutzt werden. Weiterhin konnte eine etwa 8-fache Steigerung der Transkriptionsrate für Kathepsin-D-mRNA unter Calcitriol-Stimulierung nachgewiesen werden. Eine schwerpunktmäßige Nutzung einer bestimmten Startstelle als Ursache für die gesteigerte Transkriptionsrate, wie dies etwa in Östrogen-stimulierten MCF7-Zellen der Fall ist, konnte nicht gezeigt werden

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

International audienceβ-Hexosaminidase is a soluble glycohydrolase involved in glycoconjugate degradation into lysosomes, nevertheless its localization has also been described in cytosol and plasma membrane. Recently we demonstrated the presence of Hex associated to human fibroblast plasma membrane as mature form and functionally active towards GM2 ganglioside. In this study Hex was analysed in lysosomal membrane-enriched fraction, obtained by purification from highly purified human placenta lysosomes. Results demonstrate the presence of mature Hex associated to lysosomal membrane and displaying, as the plasma membrane (PM) associated form, an acidic optimum pH. When subjected to carbonate extraction, the enzyme behave as a peripheral membrane protein, while Triton X-114 phase separation confirmed its partial hydrophilic nature, characteristics that are in common with the PM-associated Hex. Moreover 2D electrophoresis indicated a slight difference in pI of β-subunits in the membrane and the soluble forms of the lysosomal Hex. These data reveal a new aspect of the Hex biology and suggest that a fully processed membrane-associated form of Hex is translocated from the lysosomal to the plasma membrane by an as yet unknown mechanism. We present a testable hypothesis that at the cell surface Hex changes the composition of glycoconjugates that are known to be involved in intercellular communication and signaling

Endosomal targeting and secretion of lysosomal proteins in U937 cells

The present contributions on secretion and targeting of lysosomal enzymes in U937 cells are as follows: 1. The previously described strong enhancement in the secretion of lysozyme and serglycin in the presence of PMA and a minor effect on that of procathepsin D is confirmed. 2. PMA enhances the rate of the secretion of prosaposin and this may explain the induced secretion of a portion of procathepsin D, since at least a fraction of the two precursors are engaged in mutual complexes. 3. PMA is shown to induce the secretion of a high proportion of the cellular ß-hexosaminidase indicating exocytosis of an endosomal/lysosomal compartment. 4. PMA is shown to induce secretion of a portion of partially (intermediate) and fully processed (mature) forms of cathepsin D. This and the observation referred to in preceding statement indicate an effect of PMA distally to the sorting at the TGN. 5. An induction of a fusion of a distal compartment is further supported by finding Lamp-II at the plasma membrane in PMA-treated cells. 6. Tunicamycin, which inhibits N-glycosylation and secondarily the endowment of lysosomal proteins with the M6P marker, enhances the lysosomal targeting of serglycin. This effect is strongly pronounced in the presence of PMA. It can be explained by a participation of CI-MPR in the lysosomal targeting of serglycin. The receptor may have a preference for M6P ligands, that may suppress the binding of serglycin. 7. PMA does not enhance the presence of CI-MPR at the plasma membrane. 8. The effects of PMA appear to be mediated by an activation of phospholipase D and the local generation of phosphatidic acid and diacylglycerol in the membrane. It is proposed that these lipids participate in fusion and fission phenomena of different compartments and a selective enhancement of the secretion of lysosomally targeted products. 9. PMA induces a phosphorylation of several proteins in the soluble as well as the vesicular fractions of U937 cells. One of the selectively phosphorylated proteins was identified as insulin-responsive amino peptidase (IRAP). Further experiments are indicated to explore a possible role of IRAP in the selective secretory effects of PMA

Untersuchung lysosomaler Membranproteine mit dem Schwerpunkt der Charakterisierung der Acetyl-Coenzym A: a-Glucosaminid N-Acetyltransferase aus lysosomalen Membranpräparationen aus humaner Plazenta

Die Acetyl-CoA-Glucosaminid-N-Acetyltransferase ist ein lysosomales Protein, dessen Ausfall das Sanfilippo C-Syndrom (Mukopolysaccharidose Typ IIIC, OMIM 252930) bedingt. Dabei handelt es sich um eine von elf durch distinkte Enzymdefekte bedingten Mukopolysaccharidosen. Durch den Ausfall der lysosomalen Acetyltransferase kommt es zu einer Störung des Heparansulfatabbaus mit einer Ansammlung von Stoffwechselprodukten im Lysosom. Die zugrunde liegende Gensequenz wurde 2006 von Fan et al. und Hrebicek et al. beschrieben. Ein Schwerpunkt der vorliegenden Arbeit war eine Anreicherung und Solubilisierung der lysosomalen Acetyltransferase. Das Enzym wurde aus humaner Plazenta gewonnen. Nach einer Anreicherung lysosomaler Membranproteine durch Immunaffinitätsabsorption wurden die Membranen mit Detergentien solubilisiert und die extrahierten Membranproteine durch verschiedene Verfahren fraktioniert. Hierzu wurden die Extraktionsbedingungen optimiert sowie Gelfilration und Ionenaustauscherchromatographie verwendet. In den so gewonnenen Fraktionen wurde die Aktivität von Acetyltransferase bestimmt und die Fraktionen höchster Aktivität durch SDS-PAGE und 2D-Elektrofokussierung/SDS-PAGE weiter charakterisiert. Eine Isolierung markierter Acetyltransferase konnte wegen eines im Rahmen der zweidimensionalen Trennung aufgetretenen Verlusts des markierten Proteins nicht erreicht werden. Es zeigte sich jedoch, dass die Markierung wenig spezifisch war. Nach eindimensionaler Trennung wurde in dem als wahrscheinlich für die Acetyltransferase geltenden Molekulargewichtsbereichen die alkalische Phosphatase identifiziert. Auf Grund der vorliegenden Ergebnisse erscheint die Hypothese von Bame und Rome (1986) über Bildung eines acetylierten Enzymintermediats als wenig wahrscheinlich. Durch uns konnte erstmals die Assoziation der Acetyltransferase zu rafts gezeigt werden. Versuche mit -Cyclodextrin zeigten, dass die optimale Aktivität der Acetyltransferase von der Anwesenheit von Cholesterin, einem typischen Bestandteil von DRMs (detergent resistant membranes) abhängig ist. Die hier beschriebenen Trennmethoden und Solubilisierung und die erstbeschriebene Bedeutung von Cholesterin für die Aktivität der Acetyltransferase können eine Grundlage künftiger Isolierung und Rekonstitution des Membranproteins in Liposomen werden

Characterisation of lipofuscin-like lysosomal inclusion bodies from human placenta

A structural hallmark of lysosomes is heterogeneity of their contents. We describe a method for isolation of particulate materials from human placental lysosomes. After a methionine methyl ester-induced disruption of lysosomes and two density gradient centrifugations we obtained a homogeneous membrane fraction and another one enriched in particulate inclusions. The latter exhibited a yellow-brown coloration and contained bodies lacking a delimiting membrane, which were characterised by a granular pattern and high electron density. The lipofuscin-like inclusion materials were rich in tripeptidyl peptidase 1, beta-glucuronidase, acid ceramidase and apolipoprotein D and contained proteins originating from diverse subcellular localisations. Here we show that human term placenta contains lipofuscin-like lysosomal inclusions, a phenomenon usually associated with senescence in postmitotic cells. These findings imply that a simple pelleting of a lysosomal lysate is not appropriate for the isolation of lysosomal membranes, as the inclusions tend to be sedimented with the membranes. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved