The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Targeting of phosphomannosyl-deficient arylsulfatase A to lysosomes of I-cell fibroblasts

Fibroblasts from I-cell disease, a genetically-determined lysosomal storage disease, are shown to contain large amounts of phase-dense lysosomes. These lysosomes accumulated acridine orange and were specifically labeled with antibodies to arylsulfatase A. In normal skin fibroblasts the number of arylsulfatase-containing lysosomes was considerably lower. By immunocytochemistry, metabolic labeling and enzyme assay, the arylsulfatase A in I-cell fibroblasts was shown to be synthesized, stored and secreted at a level that was several-fold higher than that present in heterozygous I-cell or normal fibroblasts. Arylsulfatase A in I-cell fibroblasts differed from arylsulfatase in normal fibroblasts by the absence of endoglycosidase H-sensitive phosphorylated oligosaccharides. These findings indicate that arylsulfatase A in I-cells is targeted to lysosomes by a mechanism that does not appear to involve the phosphorylated mannose marker.     

Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.     

The isolation of lysosomes from normal rat liver by affinity chromatography

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.     

I-cell disease: deficiency of extracellular hydrolase phosphorylation

The content of ³²P-phosphorylated residues of purified extracellular N-acetyl-β-hexosaminidase obtained from the fibroblasts of I-cell disease patients was compared to that of control cells hydrolase. The analyses indicated a 60-fold decrease of the radioactivity per unit enzyme activity in the hydrolase synthesized by the patient's fibroblasts compared to the normal enzyme. Electrofocusing demonstrated again a marked decrease in the ³²P-content of the I-cell hydrolase while the control enzyme showed the presence of radioactivity in both isozymes, namely hexosaminidase A and hexosaminidase B. Most of the radioactivity could be removed from the hydrolase following incubation with alkaline phosphatase, thus indicating its phosphoester linkage.Since phosphorylated sugar residues on lysosomal enzymes function as recognition marker for their transport to the lysosomal compartment and for their specific uptake by fibroblasts, the deficiency of phosphorylated residues on the I-cell hydrolase explains the low intracellular and high extracellular lysosomal enzyme levels observed in this disease.     

Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organellers. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed in mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [³H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.     

Increased levels of sialic acid associated with a sialidase deficiency in I-cell disease (mucolipidosis II) fibroblasts.

Cultured fibroblasts from three unrelated patients with I-cell disease (mucolipidosis II) have a 3 to 4 fold increase in total sialic acid when compared to control fibroblasts. Sialic acid levels in a number of other lysosomal disorders, i.e., mucopolysaccharidosis I, II, III, VI, metachromatic leukodystrophy, G_M1 gangliosidosis, mannosidosis, Gaucher's and Sandhoff's disease are within the normal range suggesting that this is a finding specific for I-cells. Additionally, sonicates of cultured fibroblasts from controls were shown to have an acid sialidase capable of removing sialic acid from added fetuin at pH 4.2 in 0.05M acetate buffer. In contrast, I-cell fibroblasts, within the limits of the assay, lack this enzyme activity.     

Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Membranous complexes characteristic of melanocytes derived from patients with Hermansky–Pudlak syndrome type 1 are macroautophagosomal entities of the lysosomal compartment

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder resulting from mutations in a family of genes required for efficient transport of lysosomal‐related proteins from the trans‐Golgi network to a target organelle. To date, there are several genetically distinct forms of HPS. Many forms of HPS exhibit aberrant trafficking of melanosome‐targeted proteins resulting in incomplete melanosome biogenesis responsible for oculocutaneous albinism observed in patients. In HPS‐1, melanosome‐targeted proteins are localized to characteristic membranous complexes, which have morphologic similarities to macroautophagosomes. In this report, we evaluated the hypothesis that HPS‐1‐specific membranous complexes comprise a component of the lysosomal compartment of melanocytes. Using indirect immunofluorescence, an increase in co‐localization of misrouted tyrosinase with cathepsin‐L, a lysosomal cysteine protease, occurred in HPS‐1 melanocytes. In addition, ribophorin II, an integral endoplasmic reticulum protein that is also a component of macroautophagosomes, and LC3, a specific marker of macrophagosomes, demonstrated localization to membranous complexes in HPS‐1 melanocytes. At the electron microscopic level, the membranous complexes exhibited acid phosphatase activity and localization of exogenously supplied horseradish peroxidase (HRP)‐conjugated gold particles, indicating incorporation of lysosomal and endosomal components to membranous complexes, respectively. These results confirm that membranous complexes of HPS‐1 melanocytes are macroautophagosomal representatives of the lysosomal compartment.     

Five related Lebanese individuals with high plasma lysosomal hydrolases: a new defect in mannose-6-phosphate receptor recognition?

Five healthy related individuals in 3 generations of a Lebanese family have been found to have highly elevated plasma lysosomal enzyme levels inherited as a dominant Mendelian trait. The same enzymes in other extracellular fluids were within normal limits. While the pattern and extent of plasma enzyme elevation was similar to that found in mucolipidoses II and III, the physicochemical properties of the elevated enzymes were different from those of both control and I-cell disease plasma. Secretion of lysosomal hydrolases into cell media by fibroblasts from one of the individuals was increased two to seven times more than that from controls. The results suggest faulty recognition between lysosomal hydrolases and mannose-6-phosphate receptors. This could be caused by a defect either in the phosphodiesterase that normally uncovers mannose-6-phosphate hydrolase markers or in the mannose-6-phosphate receptor itself.     

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.     

The effect of monensin on beta-hexosaminidase transport in normal and I-cell fibroblasts.

The carboxylic ionophore, monensin, blocks the migration of glycoprotein-containing vesicles from the Golgi region to the plasma membrane in fibroblasts resulting in an accumulation of secretory products in the Golgi cisternae. Treatment of cultured I-cell fibroblasts with monensin (0.5 muM) decreased the abnormal excretion of beta-hexosaminidase to 40% of untreated cultures within 15 min. A corresponding intracellular accumulation of the enzyme to greater than 200% of untreated cultured by 24 h was also observed. A small intracellular accumulation and slightly enhanced excretion of beta-hexosaminidase occurred in treated normal fibroblasts cultures. The intra- and extra-cellular distribution of newly synthesized beta-hexosaminidase in both monensin-treated normal and I-cell fibroblasts were electrophoretically indistinguishable from the four bands characteristic of I-cell intracellular beta-hexosaminidase. The excreted enzyme from both cultures was found to be a low- or no-uptake form. This form of beta-hexosaminidase may have been excreted from a secondary route preceding the site of the monensin effect. The similar findings in monensin-treated normal and I-cell cultures suggest that the subcellular site of the biochemical defect in I-cell disease is at a location after the site of the monensin effect i.e. late in the Golgi region or at a post-Golgi-region location.     

Electrophoretic abnormalities of lysosomal enzymes in mucolipidosis fibroblast lines.

Electrophoretic properties of eight lysosomal hydrolases and 36 nonlysosomal enzymes were investigated in cultured fibroblasts from children with the inherited storage disease mucolipidosis II (ML II); fibroblasts from a child with a related disorder, mucolipidosis III (ML III); and two obligate heterozygous cell lines from parents of a ML II child. Cell homogenates of ML II fibroblast lines showed altered mobilities for lysosomal beta-hexosaminidase, acid phosphatase2, and alpha-mannosidase and deficient activity for the esterase-A4 and lysosomal alpha-mannosidase-B electrophoretic phenotypes. Altered mobility was also detected for the nonlysosomal enzyme adenosine deaminase-d. Deficient activities of other lysosomal enzymes were observed as previously reported. In a single ML III fibroblast line, only beta-hexosaminidase showed an abnormal electrophoretic pattern suggesting a difference between these cells and ML II fibroblasts. Thirty-five nonlysosomal enzymes associated with other cellular organelles and metabolic pathways were electrophoretically normal in all mucolipidosis cell lines. Heterozygous ML II cells showed normal expression for all enzymes. Two major patterns of altered lysosomal enzymes and adenosine deaminase were demonstrated in ML II cell lines, suggesting that at least two genetic forms of this disorder may exist. Neuraminidase treatment of ML II homogenates converted altered forms of acid phosphatase2 and adenosine deaminase-d and in two ML II lines, recovered the previously undetected lysosomal alpha-mannosidase band. These results are consistent with the mucolipidosis defect(s) being associated with abnormal post-translatinal processing of multiple lysosomal enzymes and adenosine deaminase-d.     

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

Summary The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.