Lysosomal Enzyme Precursor Research Articles

AB124. Mucolipidosis type II: clinical features and laboratories.

Background I-cell disease (Mucolipidosis II) is a rare lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-l-phosphotransferase, an enzyme that transfers phosphate groups onto oligosaccharide units of lysosomal enzyme precursors. Due to the absence of transferase activity, the common phosphomannose recognition marker of acid hydrolases is not generated, and the enzymes are not targeted to the lysosomes I. As a consequence the enzymes are secreted into the extracellular space, and high activities can be found in the serum, cerebrospinal fluid and urine of the patients, whereas inside the cells (fibroblasts) the enzyme levels are considerably reduced. Mucolipidosis is also known as I-cell disease because of the coarse granular cytoplasmic inclusions seen in cultured skin fibroblasts which are large lysosomes containing heterogeneous material.

Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation.

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.

Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.

Tilorone-induced lysosomal storage of sulphated glycosaminoglycans can be separated from tilorone-induced enhancement of lysosomal enzyme secretion

This investigation deals with a drug side-effect. The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and congeners induce lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and in cultured cells. At high tilorone concentrations, GAG storage in cultured fibroblasts was previously reported to be accompanied, and presumably caused by, disturbance of intracellular targeting of lysosomal enzyme precursors, which leads to enhanced secretion and thus loss of lysosomal enzymes. The purpose of the present study was to examine whether the GAG storage induced in cultured bovine fibroblasts by low tilorone concentrations is also accompanied by enhanced lysosomal enzyme release. Enhanced secretion of β-hexosaminidase (EC 3.2.1.52) was taken as indicating the intracellular mistargeting of lysosomal enzyme precursors. Dose-response curves were established for (a) the intracellular accumulation of 35S-GAGs and (b) the release of β-hexosaminidase after exposure (72 hr) to tilorone (1–35 μM). For positive controls, the classical lysosomotropic agents NH 4C1 (1–30 mM) and chloroquine (1–60 μM) were used. With NH 4C1, 35S-GAG storage was accompanied by enhanced enzyme release throughout the concentration range ( ec 50 at 3.3 mM for either effect). With chloroquine, low concentrations (⩽ 5 μM) caused a small increase in 35S-GAG accumulation without abnormal enzyme secretion; at higher concentrations both drug effects were produced ( ec 50 around 15 μM for either effect). With tilorone, low concentrations (⩽5 μM) caused marked 35S-GAG accumulation without enhancement of enzyme release. The ec 50 for tilorone-induced 35S-GAG storage was 3 μM, as opposed to 15 μM for enzyme release. The results indicate that GAG storage induced by low concentrations of tilorone is due to mechanisms other than mistargeting and loss of lysosomal enzymes. On the basis of previous results it may be hypothesized that tilorone and other symmetrically substituted dicationic compounds form complexes with the polyanionic GAG chains and thereby impair their enzymic degradation.

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.

Correction of Mucolipidosis III in Vitro by Gene Transfer

Mucolipidosis II (ML II, I-cell disease) and mucolipidosis III (ML III, pseudo-Hurler polydystrophy) are human autosomal recessive genetic disorders resulting from deficient UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase (GNPT) activity. Normally, this enzyme is involved in the processing of most lysosomal enzymes. Cultured fibroblasts from individuals with either disorder are deficient in a broad array of lysosomal enzymes as a result of the diminished GNPT activity. We report the correction of this phenotype by fusing transformed ML III cells generated for this study to lethally irradiated rodent cells. This method of gene transfer does not require selection for the gene of interest, animal models, nor any knowledge of the gene product except a screening method for its presence. It has generated corrected cell hybrids that contain approximately 1% hamster-derived sequences. These cell lines, which contain the hamster analogue to the human phosphotransferase gene, are useful for the molecular cloning of the gene defective in ML II and ML III.

Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.

Sertoli cells were isolated from prepubertal mice and cultured in serum-free medium to determine whether they secrete glycoproteins containing mannose 6-phosphate (M6P). Assays of the conditioned medium for lysosomal enzyme precursors, which typically bear the M6P recognition marker, indicated that Sertoli cells selectively secreted beta-N-acetylhexosaminidase and alpha-mannosidase, but not beta-glucuronidase or beta-galactosidase. Sertoli cells were labeled metabolically with [35S]methionine and the conditioned medium was fractionated on a cation-independent M6P receptor affinity column. Most of the secreted proteins did not bind to the column (peak A); however, approximately 10% of the radioactivity eluted as a low-affinity fraction (peak B), and 5-11% of the recovered cpm bound to the column and were eluted with 2.5 mM M6P (peak C). The radiolabeled proteins in each fraction were analyzed by one- and two-dimensional electrophoresis and fluorography. Two protein bands with molecular weights of 30,000 and 35,000 were present in peak B. Peak C contained at least ten M6P-containing glycoproteins with molecular weights between 30,000 and 135,000 and isoelectric points < 6.5. The 35,000-molecular-weight constituent prominent both in peaks B and C was identified as procathepsin L by immunoprecipitation with a specific antibody. When pachytene spermatocytes and round spermatids were cultured overnight in the presence of peak C glycoproteins radiolabeled with 125I, both germ cell types accumulated these Sertoli M6P-glycoproteins by a receptor-mediated process that was specifically inhibited by M6P. The Sertoli M6P-glycoproteins taken up by germ cells were processed to lower molecular weight forms. These results provide evidence that M6P receptors on the surface of spermatogenic cells endocytose secrete glycoproteins that are likely to be present in the seminiferous epithelium.

Theearly andlate processing of lysosomal enzymes: Proteolysis and compartmentation

Lysosomal enzymes are subjected to a number of modifications including carbohydrate restructuring and proteolytic maturation. Some of these reactions support lysosomal targeting, others are necessary for activation or keeping the enzyme inactive before being segregated, while still others may be adventitious. The non-segregated fraction of the enzyme is secreted and can be isolated from the medium. It is considered that the secreted lysosomal enzymes fulfill certain physiological and pathophysiological roles. By comparing the secreted and the intracellular enzymes it is possible to distinguish between the reactions that occur before and after the segregation. In this review the reactions that may influence the segregation are referred to as the early processing and those characteristic for the enzymes isolated from lysosomal compartments as the late processing. The early processing is characterized mainly by modifications of carbohydrate side chains. In the late processing, proteolytic fragmentation represents the most conspicuous changes. The review focuses on the compartmentation of the reactions and the proteolytic fragmentation of lysosomal enzyme precursors. While a plethora of proteolytic reactions are involved, our knowledge of the proteinases responsible for the particular maturation reactions remains very limited. The review points also to work with cells from patients affected with lysosomal storage disorders, which contributed to our understanding of the lysosomal apparatus.

Biosynthesis of lysosomal enzymes in cells of the End3 complementation group conditionally defective in endosomal acidification

Various aspects of lysosome biogenesis have been studied in Chinese hamster ovary (CHO) cells of the End3 complementation group (designated G.7.1 cells), which display a temperature-sensitive defect in the acidification of endosomes, but not lysosomes. In G.7.1 and normal wild-type cells grown at the permissive temperature (34 degrees C), the lysosomal enzymes alpha-glucosidase and cathepsin D were synthesized as high-molecular-weight precursors that subsequently underwent intracellular proteolytic processing to yield lower molecular weight mature forms. The mature forms of the enzymes were retained in cells, and small amounts of each precursor were secreted. However, in G.7.1 cells grown at the restrictive temperature (41 degrees C), there was a massive and inappropriate oversecretion of lysosomal enzyme precursors, which resulted in very little of the mature forms being processed and retained by the cells. This mistargeting of lysosomal enzymes was not due to an absence of phosphorylated oligosaccharides on the enzymes, nor to a defect in mannose 6-phosphate (Man6P) receptors. However, it was found that whereas G.7.1 cells had the same number of cell surface Man6P receptors at 34 degrees C and 41 degrees C, the rate of accumulation and degradation of Man6P-containing ligands was about two to three times more rapid in cells maintained at the permissive temperature. There did not appear to be any gross changes in Golgi function as the oligosaccharides of alpha-glucosidase and the Man6P receptor were processed in a similar fashion at both 34 degrees C and 41 degrees C. In addition to these studies, electron microscopic observations revealed that at 41 degrees C, G.7.1 cells accumulated inclusion-type bodies reminiscent of those found in I-cell disease fibroblasts. Thus, the biochemical and electron microscopic results on G.7.1 cells provide further evidence that acidified endosomes are important for the biogenesis of lysosomes.

Suppression of the ‘uncovering’ of mannose‐6‐phosphate residues in lysosomal enzymes in the presence of NH4Cl

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.

A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Two Glycosphingolipid Sialyltransferases Are Localized in Different Sub-Golgi Compartments in Rat Liver

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Gal beta 1----4Glc-Cer (lactosylceramide) alpha-2----3sialyltransferase; Sat-1), and CMP-NeuAc:Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4Glc-Cer (GM1 ganglioside) alpha 2----3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of cis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments.

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 Macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH 4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH 4C1 induced a 12-fold increase in TGase secretion. NH 4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-in-dependent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, α- d-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of α- d-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.

Properties of N-acetylglucosamine 1-phosphotransferase from human lymphoblasts.

Human lymphoblast and fibroblast cell lines from a patient with I-cell disease and normal individuals were characterized with respect to certain properties of UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine phosphotransferase. The enzyme isolated from normal lymphoblast and fibroblast cell lines expressed similar kinetic properties, substrate specificities and subcellular localizations. Coincident with the severe reduction of N-acetylglucosamine phosphotransferase activity in both I-cell fibroblast and lymphoblast cell lines, there was an increased secretion of several lysosomal enzymes compared to normal controls. Subsequent examination of N-acetyl-beta-D-hexosaminidase secreted by the I-cell lymphoblasts demonstrated a significant increase in adsorption of the I-cell enzyme to Ricinus communis agglutinin, a galactose-specific lectin. However, the I-cell lymphoblasts did not exhibit the significant decrease in intracellular lysosomal activities seen in I-cell fibroblasts. Our results suggest that lymphoblasts not only represent an excellent source for the purification of N-acetylglucosamine phosphotransferase, but in addition, represent a unique system for studying alternate mechanisms involved in the targeting of lysosomal enzymes.

A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough endoplasmic reticulum.

Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.

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

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

Phosphorylation of the α1,2-linked mannosylsaccharide by N-acetylglucosamine-1-phosphotransferase from fibroblasts occurs at the terminal mannose

UDP-GlcNAc: Lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase activity from normal fibroblasts was measured using methyl 2-0-(α-D-mannopyranosyl)6-deoxy-6-fluoro-α-D-mannopyranoside and methyl 2-0-(6-deoxy-6-fluoro-α-D-mannopyranosyl)α-D-mannopyranoside as acceptors. The results indicate that the phosphorylation in man α 1→2 man sequence occurs at the C-6 position of the terminal mannose residue.

UDP-N-acetylglucosamine: lysosomal enzyme precursor N-acetylglucosamine-1-phosphate transferase activities in human ovarian tumor tissue and some transformed cell lines.

Uridine diphosphate-N-acetylglucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-1-phosphate transferase, is a key enzyme involved in the intracellular targeting of lysosomal enzymes. This enzyme is elevated fourfold in primary ovarian tumor microsomes with respect to normal ovarian microsomes. This elevation is associated with significant increases in the specific activity of multiple lysosomal hydrolases, including beta-D-hexasaminidase, alpha-L-fucosidase, and beta-D-galactosidase. The activity of the phosphotransferase was also documented in several cell lines derived from human tumors. The possible role of this enzyme in tumor-associated phosphorylation is discussed.