Lysosomal Esterase Research Articles

Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene knockout (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia which was produced from L-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

Structural Engineering of Star Block Biodegradable Polymer Unimolecular Micelles for Drug Delivery in Cancer Cells.

The present investigation reports the structural engineering of biodegradable star block polycaprolactone (PCL) to tailor-make aggregated micelles and unimolecular micelles to study their effect on drug delivery aspects in cancer cell lines. Fully PCL-based star block copolymers were designed by varying the arm numbers from two to eight while keeping the arm length constant throughout. Multifunctional initiators were exploited for stepwise solvent-free melt ring-opening polymerization of ε-caprolactone and γ-substituted caprolactone to construct star block copolymers having a PCL hydrophobic core and a carboxylic PCL hydrophilic shell, respectively. A higher arm number and a higher degree of branching in star polymers facilitated the formation of unimolecular micelles as opposed to the formation of conventional multimicellar aggregates in lower arm analogues. The dense core of the unimolecular micelles enabled them to load high amounts of the anticancer drug doxorubicin (DOX, ∼12-15%) compared to the aggregated micelles (∼3-4%). The star unimolecular micelle completely degraded leading to 90% release of the loaded drug upon treatment with the lysosomal esterase enzyme in vitro. The anticancer efficacies of these DOX-loaded unimolecular micelles were tested in a breast cancer cell line (MCF-7), and their IC50 values were found to be much lower compared to those of aggregated micelles. Time-dependent cellular uptake studies by confocal microscopy revealed that unimolecular micelles were readily taken up by the cells, and enhancement of the drug concentration was observed at the intracellular level up to 36 h. The present work opens new synthetic strategies for building a next-generation biodegradable unimolecular micellar nanoplatform for drug delivery in cancer research.

A Novel Method for Carbon Nanotube Functionalization Using Immobilized Candida antarctica Lipase.

Carbon nanotubes (CNTs) have been proposed as nanovehicles for drug or antigen delivery since they can be functionalized with different biomolecules. For this purpose, different types of molecules have been chemically bonded to CNTs; however, this method has low efficiency and generates solvent waste. Candida antarctica lipase is an enzyme that, in an organic solvent, can bind a carboxylic to a hydroxyl group by esterase activity. The objective of this work was to functionalize purified CNTs with insulin as a protein model using an immobilized lipase of Candida antarctica to develop a sustainable functionalization method with high protein attachment. The functionalized CNTs were characterized by scanning electron microscope (SEM), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The enzymatic functionalization of insulin on the surface of the CNTs was found to have an efficiency of 21%, which is higher in conversion and greener than previously reported by the diimide-activated amidation method. These results suggest that enzymatic esterification is a convenient and efficient method for CNT functionalization with proteins. Moreover, this functionalization method can be used to enhance the cellular-specific release of proteins by lysosomal esterases.

Biodegradable Polymer Theranostic Fluorescent Nanoprobe for Direct Visualization and Quantitative Determination of Antimicrobial Activity.

We report a biodegradable fluorescent theranostic nanoprobe design strategy for simultaneous visualization and quantitative determination of antibacterial activity for the treatment of bacterial infections. Cationic-charged polycaprolactone (PCL) was tailor-made through ring-opening polymerization methodology, and it was self-assembled into well-defined tiny 5.0 ± 0.1 nm aqueous nanoparticles (NPs) having a zeta potential of +45 mV. Excellent bactericidal activity at 10.0 ng/mL concentration was accomplished in Gram-negative bacterium Escherichia coli (E. coli) while maintaining their nonhemolytic nature in mice red blood cells (RBC) and their nontoxic trend in wild-type mouse embryonic fibroblast cells with a selectivity index of >104. Electron microscopic studies are evident of the E. coli membrane disruption mechanism by the cationic NP with respect to their high selectivity for antibacterial activity. Anionic biomarker 8-hydroxy-pyrene-1,3,6-trisulfonic acid (HPTS) was loaded in the cationic PCL NP via electrostatic interaction to yield a new fluorescent theranostic nanoprobe to accomplish both therapeutics and diagnostics together in a single nanosystem. The theranostic NP was readily degradable by a bacteria-secreted lipase enzyme as well as by lysosomal esterase enzymes at the intracellular compartments in <12 h and support their suitability for biomedical application. In the absence of bactericidal activity, the theranostic nanoprobe functions exclusively as a biomarker to exhibit strong green-fluorescent signals in live E. coli. Once it became active, the theranostic probe induces membrane disruption on E. coli, which enabled the costaining of nuclei by red fluorescent propidium iodide. As a result, live and dead bacteria could be visualized via green and orange signals (merging of red+green), respectively, during the course of the antibacterial activity by the theranostic probe. This has enabled the development of a new image-based fluorescence assay to directly visualize and quantitatively estimate the real-time antibacterial activity. Time-dependent bactericidal activity was coupled with selective photoexcitation in a confocal microscope to demonstrate the proof-of-concept of the working principle of a theranostic probe in E. coli. This new theranostic nanoprobe creates a new platform for the simultaneous probing and treating of bacterial infections in a single nanodesign, which is very useful for a long-term impact in healthcare applications.

Synthesis of a new coumarin dye for pH independent staining of lysosomes

A novel coumarin analogue, synthesized from 2-formylcinnamic acid and resorcinol via acid catalyzed condensation and intramolecular rearrangement, is used for pH-independent staining of lysosome in live cells via lysosomal esterase mediated fluorogenic deacetylation.

Biotin-Tagged Polysaccharide Vesicular Nanocarriers for Receptor-Mediated Anticancer Drug Delivery in Cancer Cells.

Biotin-conjugated multistimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs to intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulfide and enzyme-biodegradable aliphatic ester chemical linkages, and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of <200 nm in size, which were characterized by dynamic and static light scattering, electron, and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles toward membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl)-loaded dextran vesicles exhibited stable formulation in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Redox-degradation by glutathione (GSH) showed 60% drug release, whereas lysosomal esterase enzyme enabled >98% drug release in 12 h. Confocal microscope and flow cytometry-assisted time-dependent cellular uptake studies revealed that the biotin-receptors overexpressed in cervical cancer cells (HeLa) exhibited larger drug accumulation through the receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pretreatment in cancer cells and energy-suppressed cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptor-overexpressed cervical cancer cells.

Effects of Glutathione on Hydrolytic Enzyme Activity in the Mouse Hepatocytes.

In this study, the effect of glutathione (GSH) on the activity of hydrolytic enzymes of lysosomal, microsomal, and cytosolic fractions was studied in the mouse hepatocytes. The experiments involved 30 Swiss male mice, divided into the experimental and control groups of 15 mice each. The former group received injections of 12μL/g of GSH solution at a dose of 100μg/g body weight, whereas the latter received 12μL/g of physiological saline, all given intraperitoneally daily for 7days. Then, fragments of liver tissue were collected from the euthanized animals and processed to obtain lysosomal, microsomal, and cytosolic fractions of hepatocytes. The activity of the following enzymes was investigated in vitro: β-glucuronidase, β-galactosidase, β-glucosidase, N-acetyl-hexosaminidase, lysosomal esterase and lipase, acid phosphatase, cathepsin D and L, leucine aminopeptidase, and alanine aminopeptidase. We found that GSH, administered in vivo in the mouse, in the main (73% cases), increased the in vitro activity of the majority enzymes abovementioned, although the effect was somehow variable, depending on the fraction of hepatocytes and the type of enzyme. The findings imply that GSH supplementation may intensify the rate of cellular hydrolytic degradation, i.e., the rate of disposal by the cell of unwanted materials.

Dual Functional Nanocarrier for Cellular Imaging and Drug Delivery in Cancer Cells Based on π-Conjugated Core and Biodegradable Polymer Arms.

Multipurpose polymer nanoscaffolds for cellular imaging and delivery of anticancer drug are urgently required for the cancer therapy. The present investigation reports a new polymer drug delivery concept based on biodegradable polycaprolactone (PCL) and highly luminescent π-conjugated fluorophore as dual functional nanocarrier for cellular imaging and delivery vehicles for anticancer drug to cancer cells. To accomplish this goal, a new substituted caprolactone monomer was designed, and it was subjected to ring opening polymerization using a blue luminescent bishydroxyloligo-phenylenevinylene (OPV) fluorophore as an initiator. A series of A-B-A triblock copolymer building blocks with a fixed OPV π-core and variable chain biodegradable PCL arm length were tailor-made. These triblocks self-assembled in organic solvents to produce well-defined helical nanofibers, whereas in water they produced spherical nanoparticles (size ∼150 nm) with blue luminescence. The hydrophobic pocket of the polymer nanoparticle was found to be an efficient host for loading water insoluble anticancer drug such as doxorubicin (DOX). The photophysical studies revealed that there was no cross-talking between the OPV and DOX chromophores, and their optical purity was retained in the nanoparticle assembly for cellular imaging. In vitro studies revealed that the biodegradable PCL arm was susceptible to enzymatic cleavage at the intracellular lysosomal esterase under physiological conditions to release the loaded drugs. The nascent nanoparticles were found to be nontoxic to cancer cells, whereas the DOX-loaded nanoparticles accomplished more than 80% killing in HeLa cells. Confocal microscopic analysis confirmed the cell penetrating ability of the blue luminescent polymer nanoparticles and their accumulation preferably in the cytoplasm. The DOX loaded red luminescent polymer nanoparticles were also taken up by the cells, and the drug was found to be accumulated at the perinuclear environment. The new nanocarrier approach reported in the present manuscript accomplishes both cellular imaging and delivering drugs to intracellular compartments in a single polymer system. The present investigation is one of the first examples to demonstrate the dual functional biodegradable luminescence nanocarrier concept in the literature, and the studies established this proof-of-concept in cellular imaging and drug delivery in cancer cells.

A fluorescent light-up probe with "AIE + ESIPT" characteristics for specific detection of lysosomal esterase.

We report a fluorescent light-up probe AIE-Lyso-1 for in situ visualization of lysosomal esterase activity. The probe is based on a salicyladazine fluorogen, which is conjugated with esterase reactive acetoxyl groups and lysosome-targeting morpholine moieties. The probe has characteristics of both aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT), which shows significant advantages, such as lysosome-specific targeting, no self-quenching at high concentration, excellent light-up ratio, large Stokes shift, low cytotoxicity, and high specificity to esterase. It has also been used for in situ monitoring of lysosomal esterase activity and tracking lysosomal movements in living cells, which has great potential for the diagnosis of Wolman disease caused by deficiency of lysosomal esterase.

Autophagy in MHC Class II Presentation: Sampling from Within

MHC class II molecules usually present exogenous antigens, but peptidome analyses have also identified many antigens from cytosolic or nuclear sources. In this issue of Immunity, Schmid et al. show that MHC class II molecules can present these through autophagosomes.

The influence of selection on reaction to stress in mice. IX. Effect of dietary protein level on activity of lysosomal enzymes in liver and kidney

SummaryThe studies were carried out on 48 8‐week‐old Swiss male mice. The animals were divided into those selected for body weight gain (experimental) and an unselected (control) group. Twelve selected and unselected mice received a high protein diet (40%) during 14 days while the remaining 12 selected and 12 controls received a low protein feed (10%). In the lysosomal fraction of the liver and kidney, the activities of alanyl aminopeptidase, leucyl‐aminopeptidase, cathepsin D, L, acid phosphatase, lysosomal esterase, lysosomal lipase, beta‐galactosidase, beta‐glucosidase, beta‐glucuronidase, N‐acetyl‐beta‐glucosaminidase were measured. The results obtained suggest that the different dietary protein levels (10 and 40%) and selection changed the activity of lysosomal enzymes studied in the liver and kidney of experimental mice.

Influence of microcystine–YR and nodularin on the activity of some glucosidases in mouse liver

Microcystine–YR (20 ug/kg bodyweight) and 8 ug/kg bodyweight of nodularin were intraperitoneally injected to 90 female Swiss mice. After 15, 30, 60 min and 24 h the changes were observed in the activity of some glucosidases in the complete cell homogenate and in the lysosomal, microsomal and cytosol fraction. Significant differences were connected with the time after administration of poison and with the cellular fraction. Nodularin induces the activity of β- d-glucuronidase, α-glucosidase, lysosomal esterase and N-acetyl-glucosaminidase and influenced on the labilisation of endoplasmatic reticulum membranes. Microcystine–YR inhibited the biosynthesis of glucosidases and revealed a destructive effect on membranes of lysosomes and endoplasmatic reticulum.

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells: accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (<40–50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.—Brown, A. J., E. L. Mander, I. C. Gelissen, L. Kritharides, R. T. Dean, and W. Jessup. Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells: accumulation of oxidized esters in lysosomes.

The influence of selection on reaction to stress in mice.VII. Activity of some lysosomal enzymes in the muscle, spleen, kidney and liver

The experiment was carried out on 30 56‐day old mice selected during 12 generations for feed efficiency and 30 unselected mice. Measurements were made of beta‐glucuronidase; beta‐galactosidase; beta‐glucosidase; N‐acetyl‐beta‐glucosaminidase; acid phosphatase; alanyl aminopeptidase; cathepsin D; lysosomal esterase and lysosomal lipase. The results suggest that the selected animals had higher activities of the lysosomal enzymes in comparison with unselected ones.

Effect of Ethanol Administration on Activities of Some Lysosomal Hydrolases in the Mouse

1. The studies were carried on 98 random 6-week-old male mice. The mice were divided into control and experimental groups. The experimental animals were given 10% and 20% ethanol solution daily for 7, 14 and 28 days. 2. In the lysosomal fractions of the liver, kidneys, muscle and brain, the activities of β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, alanyl-aminopeptidase, leucyl-aminopeptidase, lysosomal esterase and lysosomal lipase were affected. 3. The changes in enzyme activities in the investigated tissue were related to the time and concentration of the administered ethanol.

Characterization of the egg vesicular components in the seaweed, Fucus serratus L. (Fucales, Phaeophyta), using enzyme histochemistry and vital staining: the search for a lysosome-like body.

Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles.

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

Vinblastine-induced autophagy in mouse liver

The vinblastine effect on the size of autophagy in a mouse liver cell was studied. The activity of cathepsin D and L, lysosomal esterase, N-acetylo-beta-glucosaminidase, beta-galactosidase, beta-glucuronidase, acid phosphatase was determined. In an isolated lysosomal fraction, it was found that vinblastine caused an increase of protein degradation of mouse hepatocytes and this action was dependent on dose and time of exposure. It was related to an increase in lysosomal compartment.

METABOLICALLY CONVERTIBLE LIPOPHILIC DERIVATIVES OF pH‐SENSITIVE AMPHIPATHIC PHOTOSENSITIZERS

We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetically defined lysosomal acetylesterase EC 3.1.1.6 in the cauda epididymidis of mouse, rat, and man.

After inhibition by bis-p-nitrophenyl phosphate and subsequent staining for esterase using naphthol AS-D acetate as the substrate, a strong lysosomal esterase was demonstrated in the cauda epididymidis of mouse, rat, and man. Owing to its behaviour towards the classifying inhibitors eserine, diisopropyl fluorophosphate, bis-p-nitrophenyl phosphate, and p-chloromercuriphenylsulphonate, this lysosomal esterase was shown to be an acetylesterase (EC 3.1.1.6). Control experiments involving isoelectric focusing revealed that this acetylesterase was identical with the genetically defined homologues ES-17, ES-6, and ES-A4 in mouse, rat, and man, respectively.