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.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

Glycoconjugate markers of joint diseases

A number of different types of glycoconjugate are found associated with joint tissue and fluids, comprising glycoproteins, glycolipids and glycosaminoglycans. Oligosaccharide chains of glycoconjugates are degraded by exoglycosidases, and the dominant exoglycosidase found in human blood, synovial fluid, the synovial membrane and chondrocytes of articular cartilage is HEX (N-acetyl-β-hexosaminidase). HEX is localized mostly intracellularly in synovial cells. Serum activity of HEX may be used to monitor the course and efficiency of treatment of Lyme arthritis, and activity of HEX, above 10 μkat/kg of protein in the synovial fluid, suggests rheumatoid disease. There is a shortage of HEX inhibitors able to penetrate synoviocytes, so the development of drugs which inhibit synthesis and/or the activity of HEX will be a promising field for future investigations.     

Free amino acids in the early cleavage stages of the rabbit egg

In the first half of gestation, mouse yolk sac contains levels of N-acetyl-β-hexosaminidase activity and a ratio of N-acetyl-β-hexosaminidase: β-glucuronidase much higher than those of other embryonic tissues or the decidua. The properties of the yolk sac enzyme are very similar to those in the other tissues, suggesting that the observed difference in activity is quantitative rather than qualitative. In blastocyst cultures, the vesicular growths derived from the inner cell mass possess patterns of N-acetyl-β-hexosaminidase activity similar to yolk sac in vivo, but different from trophoblast cells developing in the same cultures. It is therefore proposed that the vesicular growths contain at least some differentiated yolk sac cells.     

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.     

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.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Intercellular exchange of lysosomal enzymes: enzyme assays in single human fibroblasts after co-cultivation.

Intercellular exchange of N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff's disease an increase of activity of N-acetyl-β-D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glycogenosis II and GM1-gangliosidosis, no indication was found for intercellular transfer of acid α-glucosidase and β-galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes.     

Salivary secretions from the ectoparasitic wasp, Eulophus pennicornis contain hydrolases, and kill host hemocytes by apoptosis

The current work demonstrates that larvae of the ectoparasitic wasp, Eulophus pennicornis, produce salivary secretions (Ep S) that contain a variety of hydrolases. This includes medium to high levels of N-acetyl-β-glucosaminidase activity, relatively low levels of esterase (C 4) and lipase (C 14) activity, and trace levels of esterase lipase (C 8), acid phosphatise, β-galactosidase, β-glucuronidase, and α-glucosidase activity. In addition, in vitro monolayer assays indicate that both a 4 h and an 18 h incubation of hemocytes from host Lacanobia oleracea larvae in Ep S significantly increases (P < 0.001) the percentage of cells dying by apoptosis compared to the controls. Moreover, an 18 h incubation of hemocytes in Ep S (but not a 4 h incubation), also significantly increased the percentage of dead cells detected using a trypan-blue exclusion assay. The role of ectoparasitoid salivary secretions in conditioning the host by disabling hemocyte-mediated wound healing responses, and providing food for the developing wasp larvae is discussed.     

Marqueurs du tractus génital et activités glycosidasiques séminales

Seminal liquefaction corresponds to a proteolysis of glycoproteins and could also result in a lysis of linked glycans involving glycosidases. It is well known that α-1-4 glucosidase is a real epipidymal marker. The aim of this study is to point out new markers among the glycosidases and especially β-D-mannosidase, α-L-fucosidase and N-acetyl-β-D-glucosaminidase. There is no correlation between the three enzymatic activities and the sperm count. Moreover in case of azoospermia enzymatic activities were always present. The variations observed are both quantitative (β-MAN activity is significantly decreased) and qualitative (modifications of the relative proportions of the different enzymatic forms) according to the etiology of azoospermia.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

The measurement of N-acetyl-β-glucosaminidase activity upon β-MAGA was carried out in order to survey oligosaccharidase fraction in the chitinolytic enzymes of Aspergillus niger. N-Acetyl-β-glucosaminidase was purified in parallel with chitobiase activity, being separated from chitinase activity, and some properties of the enzyme in the hydrolysis of β-MAGA and DACE were investigated. The enzyme hydrolysed more rapidly S-glueosaminidie bonds in DACE than that in β-MAGA, but did not decompose α-MAGA.     

Binding of lysosomal hydrolases by the membranes of rat liver lysosomes: Evidence for phosphomannosyl recognition markers

Most of the soluble hydrolase activity of broken lysosomes was found to be bound to lysosomal membranes. However, this soluble activity could be released from the membranes by the addition of sugar phosphates. Mannose-6-phosphate was found to displace N-acetyl-β-D-glucosaminidase (NAβGase) from the membrane in a concentration dependent manner. In addition, fructose-6-phosphate and AMP were also effective. The binding of β-glucuronidase was similarly affected by sugar phosphates. The glycosyl specificity of the lysosomal membrane receptor appears to be similar to that of the plasma membrane receptor in cultured fibroblasts, as previously reported (Kaplan, A., Achord, D. T., and Sly, W. S. (1977) Proc. Natl. Acad. Sci. $\text{74}$, 2026–2030). These results indicate that phosphomannosyl receptors for the lysosomal hydrolases may exist in the lysosomal membrane as well as in the plasma membrane.     

Cell specificity in the developmental regulation of acid hydrolases by temporal genes

The cell specificity of expression of three distinct trans acting temporal gene systems determining the developmental control of α-galactosidase, β-galactosidase and β-glucuronidase was tested in mouse liver. For α-galactosidase and β-galactosidase, expression was limited to hepatocytes; no effect was seen in nonhepatocytes. For β-glucuronidase the data suggest that expression of the Gus-t temporal locus is also limited to hepatocytes, and that the smaller enzyme reduction seen in nonhepatocytes of some strains is due to a separate systemic regulatory locus that is also present in the [Gus] gene complex. We conclude that the temporal gene-determined timing mechanisms initiating switches in rates of enzyme synthesis are intrinsic to the cells themselves and are not communicated to adjacent cells. This conclusion applies to the temporal locus for β-glucuronidase that is proximate to its structural gene as well as those for α-galactosidase and β-galactosidase that are distant from the structural genes that they regulate.     

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kg_bodyweight h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.     

Inactivation by chloroquine of alpha-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

The distribution of hydrolytic enzyme activities in human fibroblast cultures and their intercellular transfer.

Both N-acetyl-β-D-glucosaminidase A and B (EC 3.2.1.30) are continuously secreted by normal cultured fibroblasts and can be taken up by deficient Sandhoff cells without cellular contact. The absence of intercellular transfer of β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) in cocultivations of normal and deficient fibroblasts is accompanied by very low extracellular activities of these enzymes in cultures of normal fibroblasts. For each of the hydrolases tested an appreciable amount of activity was found in the “pericellular” fraction. N-acetyl-β-D-glucosaminidase which has been taken up by deficient Sandhoff cells has an intracellular half-life of 6 days. The ingested intracellular enzyme, which is presumably localized in the lysosomes, is partly transferred to the pericellular fraction, and to the extracellular fraction. The results are discussed in relation to the secretion-recapture model proposed by Hickman and Neufeld.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism

The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE^?/?) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE^?/? mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, β-glucuronidase, N-acetyl-β-d-hexosaminidase, β-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.