Starvation enhances lipoprotein lipase activity in the liver of the newborn rat

Using metabolic labelling and sucrose density fractionation we compared the synthesis of lysozyme and lysosomal enzymes in human monocytic U937 cells. In pulse-chase experiments in sucrose density gradients, the intracellular radioactively labelled lysozyme distributed similarly to cathepsin D and beta-hexosaminidase. With the aid of immunochemical detection in Western blots, the steady-state distribution of lysozyme was found to be slightly different from that of beta-hexosaminidase; relatively more lysozyme was present in fractions sedimenting between lysosomes and the Golgi apparatus. The observed distribution of the lysozyme antigen with a prominent peak in the lysosomal fraction was in striking contrast to the broad distribution of the lysozyme activity. The difference was explained by a bias in the determination of the activity of lysozyme by the 'lysoplate' diffusion assay.     

Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients

Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the β-subunit of β-hexosaminidase, that result in β-hexosaminidase A (αβ) and β-hexosaminidase B (ββ) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal–lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal–lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.     

Glycohydrolases β-hexosaminidase and β-galactosidase are associated with lipid microdomains of Jurkat T-lymphocytes

Growing evidence suggests the presence of active lysosomal enzymes in extra-lysosomal compartments, such as the plasma membrane. Although in the past little attention was paid to glycohydrolases acting on cellular compartments different from lysosomes, there is now increasing interest on plasma membrane-associated glycohydrolases because they should be involved, together with glycosyltransferases, in glycosphingolipids oligosaccharide modification processes regulating cell-to-cell and/or cell-environment interactions in both physiological and pathological conditions. Starting from the previous evidence of the presence of β-hexosaminidase and β-galactosidase at the plasma membrane of cultured fibroblasts, we here investigated the association of these glycohydrolases with lipid microdomains of Jurkat T-lymphocytes. Monosialoganglioside GM3 represents the major glycosphingolipid constituent of T-cell plasma membrane and its amount largely increases after T-cell stimulation. β-hexosaminidase and β-galactosidase cleave specific β-linked terminal residues from a wide range of glycoconjugates and in particular are involved in the stepwise degradation of GM1 to GM3 ganglioside. Here we demonstrated that fully processed plasma membrane-associated β-hexosaminidase and β-galactosidase co-distribute with the lipid microdomain markers and co-immunoprecipitate with the signalling protein lck in Jurkat T-cell. Furthermore, Jurkat cell stimulation up-regulates the expression and activity of lysosomal β-hexosaminidase and β-galactosidase and increases their targeting to lipid microdomains. The non-random distribution of plasma membrane-associated β-hexosaminidase and β-galactosidase and their localization within lipid microdomains, suggest a role of these enzymes in the local reorganization of glycosphingolipid-based signalling units.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

High frequency of-β-hexosaminidase deficiency in lymphoblastoid cell lines

The activity of seven lysosomal enzymes was determined in 25 lymphoblastoid cell lines. These lines included normal controls transformed with Epstein-Barr virus, Burkitt's lymphomas and other lymphomas with or without EBV genome.Four lines were deficient in total β-hexosaminidase activity. The deficiency was as severe as that of the variant O (Sandhoff's disease) of clinical β-hexosaminidase deficiency. The electrophoretic pattern was also similar to that observed in Sandhoff's disease.The possible mechanisms explaining the high frequency of β-hexosaminidase deficiency in lymphoblastoid cell lines are discussed.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, β-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A₁ and A₂ which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl₂. Homogenates of BCG-induced macrophages had specific activities of β-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 10⁷ cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.     

Exoglycosidase markers of diseases

Exoglycosidases are hydrolases involved in lysosomal degradation of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). In tissues and body fluids, a higher exoglycosidase specific activity is found in N-acetyl-β-hexosaminidase, than β-glucuronidase, α-L-fucosidase, β-galactosidase, α-mannosidase and α-glucosidase. Determination of exoglycosidases (especially N-acetyl-β-hexosaminidase and β-glucuronidase) in body fluids could be an inexpensive, easy to perform and sensitive test for pathological evaluation, as well as in screening and monitoring many diseases, including alcohol abuse, risk of arteriosclerosis, bacterial infections (e.g. Lyme borreliosis), chronic inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis, asthma, autoimmune hepatitis and primary biliary cirrhosis, as well as cancers.     

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal–lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimer's disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimer's disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimer's disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimer's disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimer's disease.     

Differential inhibition of beta-hexosaminidase A and beta-hexosaminidase B by suramin

The trypanocidal drug suramin is a potent inhibitor of β-hexosaminidase A with a K_i of about 4.5 μM, and to a lesser extent of β-hexosaminidase B (K_i 31.5 μM). β-Hexosaminidase B remained active in the presence of 1.0 mM suramin whereas the activity of β-hexosaminidase A was completely inhibited at 0.1 mM.     

Effects of Arachidonic Acid on the Lysosomal Ion Permeability and Osmotic Stability

In this study, we investigated the effects of arachidonic acid, a PLA₂-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K⁺/H⁺ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Mouse liver lysosomes contain enzymatically active processed forms of Hyal-1

It has long been known that liver lysosomes contain an endoglycosidase activity able to degrade the high molecular mass glycosaminoglycan hyaluronic acid (HA). The identification and cloning of a hyaluronidase with an acidic pH optimum, Hyal-1, suggested it might be responsible for this activity. However, we previously reported that this hydrolase could only be detected in pre-lysosomal compartments of the mouse liver using a zymography technique that allows the detection of Hyal-1 activity after SDS–PAGE (“renatured protein zymography”). Present work reveals that the activity highlighted by this technique belongs to a precursor form of Hyal-1 and that the lysosomal HA endoglycosidase activity of the mouse liver is accounted for by a proteolytically processed form of Hyal-1 that can only be detected using “native protein zymography”. Indeed, the distribution of this form follows the distribution of β-galactosidase, a well-established lysosomal marker, after fractionation of the mouse liver in a linear sucrose density gradient. In addition, both activities shift toward the lower density region of the gradient when a specific decrease of the lysosomal density is induced by Triton WR-1339 injection. The fact that only native protein zymography but not renatured protein zymography is able to detect Hyal-1 activity in lysosomes points to a non-covalent association of Hyal-1 proteolytic fragments or the existence of closely linked partners supporting Hyal-1 enzymatic activity. The knockdown of Hyal-1 results in an 80% decrease of total acid hyaluronidase activity in the mouse liver, confirming that Hyal-1 is a key actor of HA catabolism in this organ.     

Hyaluronidase 1 and β-hexosaminidase have redundant functions in hyaluronan and chondroitin sulfate degradation

Hyaluronan (HA), a member of the glycosaminoglycan (GAG) family, is a critical component of the extracellular matrix. A model for HA degradation that invokes the activity of both hyaluronidases and exoglycosidases has been advanced. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to HA breakdown. Herein, we used mouse models to investigate the contributions of the endoglycosidase HYAL1 and the exoglycosidase β-hexosaminidase to the lysosomal degradation of HA. We employed histochemistry and fluorophore-assisted carbohydrate electrophoresis to determine the degree of HA accumulation in mice deficient in one or both enzyme activities. Global HA accumulation was present in mice deficient in both enzymes, with the highest levels found in the lymph node and liver. Chondroitin, a GAG similar in structure to HA, also broadly accumulated in mice deficient in both enzymes. Accumulation of chondroitin sulfate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulfate breakdown. Extensive accumulation of HA and chondroitin when both enzymes are lacking was not observed in mice deficient in only one of these enzymes, suggesting that HYAL1 and β-hexosaminidase are functionally redundant in HA and chondroitin breakdown. Furthermore, accumulation of sulfated chondroitin in tissues provides in vivo evidence that both HYAL1 and β-hexosaminidase cleave chondroitin sulfate, but it is a preferred substrate for β-hexosaminidase. These studies provide in vivo evidence to support and extend existing knowledge of GAG breakdown.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.     

Phosphorylation of GIT1 tyrosine 321 is required for association with FAK at focal adhesions and for PDGF-activated migration of osteoblasts

Lysosomes are not only degrading organelles but also involved in other critical cellular processes. In addition, active lysosomal glycohydrolases have been detected in an extra-lysosomal compartment: the presence of glycohydrolases on the plasma membrane (PM) has been widely demonstrated, and a possible role on the modification of the cell surface glycosphingolipids (GSL) participating in the modulation of cell functions such as cell-to-cell interactions and signal transduction pathways has been proposed. On this basis, the coordinated expression of lysosomal glycohydrolases and their translocation to the PM appear to be crucial for many cellular events. In this paper, we report evidence for the existence of a coordinated mechanism regulating the expression/activity of both lysosomal and PM-associated glycohydrolases. We show that the over-expression of the acidic glycohydrolase β-hexosaminidase α-subunit in mouse NIH/3T3 fibroblasts induces the increased expression of the Hex β-subunit necessary to form the active isoenzyme dimers as well as of other glycohydrolases participating in the GSL catabolism, such as β-galactosidase and β-glucocerebrosidase. More interestingly, this regulatory effect was also extended to the PM-associated hydrolases. In addition, transfected cells displayed a rearrangement of the GSL expression pattern that cannot be simply explained by the increased activity of a single enzyme. These observations clearly indicate that the expression level of metabolically related glycohydrolases is regulated in a coordinated manner and this regulation mechanism also involves the PM-associated isoforms.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Mechanism of Lysophosphatidylcholine-Induced Lysosome Destabilization

Lysosomal destabilization is critical for the organelle and living cells. Phospholipase A₂ (PLA₂) was shown to be able to destabilize lysosomes under some conditions. By what mechanism the enzyme affects lysosomal stability is not fully studied. In this study, we investigated the effects of lysophosphatidylcholine (lysoPC), a PLA₂-produced lipid metabolite, on lysosomal ion permeability, osmotic sensitivity and stability. By measuring lysosomal β-hexosaminidase free activity, membrane potential, proton leakage and their enzyme latency loss in hypotonic sucrose medium, we established that lysoPC could increase the lysosomal permeability to both potassium ions and protons and enhance lysosomal osmotic sensitivity. These changes in lysosomal membrane properties promoted entry of potassium ions into lysosomes via K⁺/H⁺ exchange. The resultant osmotic imbalance across the membranes led to losses of lysosomal integrity. The enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. These results suggest that lysoPC may play a key role in PLA₂-induced lysosomal destabilization.