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

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking

Congenital disorder of glycosylation Ib (CDG-Ib)

Congenital Disorders of Glycosylation (CDG) sind eine Gruppe hereditärer Störungen der N-Glykosylierung. Gemeinsames Charakteristikum dieser Multisystemerkrankungen ist eine psychomotorische Retardierung. Eine Ausnahme bildet das im Rahmen dieser Arbeit entdeckte CDG-Ib. Hauptmerkmal dieser rezessiv vererbten Erkrankung ist eine exsudative Enteropathie. Eine mentale Retardierung tritt nicht auf. Neben dem Nachweis einer signifikant reduzierten Aktivität der Phosphomannose Isomerase (PMI), die zum vollständigen Fehlen von proteingebundenen Oligosacchariden führt, konnte gezeigt werden, dass die diätetische Substitution von Mannose eine wirksame Therapie darstellt. Weiterhin konnten 22 Fälle mit Phosphomannomutase (PMM) Defizienz (CDG-Ia) und 13 bislang ungeklärte CDG diagnostiziert werden. Der Vergleich mit Galaktosämie, hereditärer Fructoseintoleranz, Alkoholabusus und verschiedenen hepatischen Erkrankungen zeigte zum Teil den CDG ähnliche Elektrophoresemuster von Serumtransferrin

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Crude chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (Percoll) gradients. The chromaffin vesicle fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicle fractions, but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicles during the biogenesis of lysosomes but acetylcholinesterase does

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