Biosynthesis, Processing, and Intracellular Transport of GM2 Activator Protein in Human Epidermal Keratinocytes
THE LYSOSOMAL TARGETING OF THE GM2 ACTIVATOR IS INDEPENDENT OF A MANNOSE-6-PHOSPHATE SIGNAL*

(Received for publication, September 25, 1996, and in revised form, November 19, 1996)

Gereon J. Glombitza Dagger , Elisabeth Becker Dagger , Hans Wilhelm Kaiser § and Konrad Sandhoff Dagger

From the Dagger  Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Federal Republic of Germany and § Hautklinik der Universitätskliniken Bonn, D-53127 Bonn, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The processing, intracellular transport, and endocytosis of the GM2 activator protein (GM2AP), an essential cofactor of beta -hexosaminidase A for the degradation of ganglioside GM2, was investigated in human epidermal keratinocytes. The GM2AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24-27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the GM2AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated GM2AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated GM2AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of GM2AP exists in cultured human keratinocytes.


INTRODUCTION

The lysosomal degradation of cellular glycosphingolipids with short oligosaccharide chains requires the presence of nonenzymatic cofactors, the glycosphingolipid activator proteins (reviewed in Refs. 1 and 2). Five different activator proteins have been discovered to date. Four of them (sphingolipid activator proteins or saposins A-D) are proteolytically generated from a single precursor (3-5). The fifth activator, the GM21 activator protein (GM2AP), is the product of a separate gene (6). It is an essential cofactor to beta -hexosaminidase A in the degradation of GM2 to GM3 (1). Its physiological significance became apparent when functional defects in GM2AP were found to be the underlying cause for the AB variant of GM2 gangliosidosis (7). This group of lysosomal storage diseases is characterized by a pronounced accumulation of undegraded GM2 and related glycolipids in the neuronal tissue of affected patients (reviewed in Ref. 8). GM2AP has been fully sequenced on the protein level; its cDNA and genomic structure have been characterized; its human gene has been localized to chromosome 5, and a processed pseudogene has been localized to chromosome 3 (reviewed in Ref. 1); and its mode of action is a subject of current debate (2, 9).

On its biosynthesis and transport, limited information was available from an earlier study in human fibroblasts (10), but many aspects of its intracellular transport remained unclear, since the expression level of GM2AP is extremely low in this cell type. Although the targeting of soluble lysosomal proteins usually involves the mannose-6-phosphate receptor system (reviewed in Ref. 11), it was found that recombinant, nonglycosylated GM2AP is effectively endocytosed by AB variant fibroblasts (12). Thus, at least the endocytosis of GM2AP can occur independently of M6P residues and this raises the question, how far its intracellular transport would depend on the M6P pathway. The existence of M6P-independent targeting mechanisms has been known for a long time. Lysosomal enzymes are hypersecreted by many cell types from I-cell disease patients, since a defect in phosphotransferase activity prevents proper phosphorylation of mannose residues in this disease. However, almost normal levels of lysosomal enzymes were found in some tissues from these patients (13), indicating compensation of the phosphotransferase defect by an alternative targeting mechanism in some cell types. M6P-independent targeting has also been described for cathepsin D in HepG2 cells (14), breast cancer cells (15), and I-cell disease lymphoblasts (16), as well as for beta -aspartylglucosaminidase (17) and alpha -glucosidase (18) in transfected COS1 cells. For cathepsin D, this type of targeting seems to involve a transient, M6P-independent association of the precursor with intracellular membranes (15, 19). Cathepsin D has been found to be associated with sphingolipid activator protein precursor during early stages of transport, and it was proposed that such an intermolecular association of lysosomal enzyme precursors might be one of the essential features of M6P-independent transport (20). It has also been suggested that the targeting signal of cathepsin D might reside in a peptide determinant in its carboxyl-terminal region (16).

To define the intracellular transport of GM2AP with particular attention to its M6P dependence, we were in need of a physiological cell culture system allowing pulse-chase analysis with reasonable amounts of radioactive label in sufficiently short pulse times. Although no data were yet available on the biosynthesis and processing of lysosomal proteins in human epidermal keratinocytes (hEKs), we decided to choose this cell type for our study, since in hEKs, GM2AP biosynthesis is 5-fold enhanced over human fibroblasts.2

With the present study, we established the biosynthesis and processing of GM2AP in hEKs and we will show that an M6P-independent targeting pathway exists for GM2AP in this cell type, which cannot be used by the precursors of cathepsins D and L. In this way, we also give first examples of lysosomal enzyme processing in hEK. We will also show, that GM2AP endocytosis is independent of known signals for N-glycan receptor-mediated endocytosis in hEK.


EXPERIMENTAL PROCEDURES

Reagents

The following reagents were purchased from commercial sources: MCDB 153 medium base, keratinocyte growth medium supplements, Dulbecco's modified Eagle's medium, N-acetylalanylglutamine (Biochrom, Berlin, Germany), cysteine- and methionine-free MCDB 153 medium base (Cytogen, Lohmar, Germany), bovine pituitary extract (Beckton Dickinson, Bedford, MA), porcine insulin, epidermal growth factor, brefeldin A, tunicamycin, yeast mannan, and protein G-Sepharose fast flow (Sigma), [35S]cysteine and [33P]orthophosphate (Amersham Corp.), [35S]sulfate (ICN, Meckenheim, Germany), and beta -endo-N-acetylglucosaminidase H (endo H) and peptide-N-glycanase F (PNGase F; New England Biolabs, Schwalbach, Germany). All other reagents were of the highest purity grade commercially available and obtained from Sigma, ICN, Merck (Darmstadt, Germany), and Serva (Heidelberg, Germany). Asialo-orosomucoid (desialylated alpha 1-acid glycoprotein, human) was a kind gift of Dr. G. Schwarzmann (Institut fur Organische Chemie und Biochemie, Universität Bonn, Bonn, Germany).

Cell Culture

Epidermal keratinocytes from human foreskin were obtained according to the method of Rheinwald and Green (21). They were cultured at 0.1 mM Ca2+ in MCDB 153 supplemented as described (22), with N-acetyl-alanyl-glutamine substituting for glutamine. At approximately 70% confluency, the cells were split in a 1:5 ratio, resulting in seeding densities of 2 × 104 cells/cm2. Passaging was repeated every 4-6 days. In the third or fourth passage, cells from a single donor were used for experiments.

Antibodies

A goat antiserum raised against recombinant GM2AP (23) was used for all experiments. Sheep anticathepsins L or D were commercially available (BioAss, Schwalbach, Germany); goat anti-beta -hexosaminidase beta -chain was a kind gift of Dr. R. Proia (NIDDK, National Institutes of Health, Bethesda, MD).

Metabolic Labeling

Epidermal keratinocytes grown in 60-mm dishes were kept in 1.5 ml of medium lacking methionine and cysteine for 2 h. When inhibitors were applied, they were already present during this starvation period. All inhibitors were added from 1000-fold-concentrated stock solutions in ME2SO. Half of the medium was then removed, and the cells were labeled with [35S]cysteine (5.55 MBq; specific activity, >37 TBq/mmol) for 60 min. After the pulse period, the cells were washed twice with phosphate-buffered saline, and the chase was initiated by addition of 1.8 ml of complete medium. The media were saved, and the cells were extracted. Phosphate labeling was performed analogously, except that the labeling medium was phosphate deficient. 18.5 MBq of [33P]orthophosphate (specific activity, >111 TBq/mmol) were used in a pulse of 5 h.

Endocytosis Experiments

Epidermal keratinocytes grown in 75-cm2 flasks were starved for 2 h in the presence of 10 mM NH4Cl and labeled with 22.2 MBq of [35S]cysteine in 4 ml of medium containing 10 mM NH4Cl for 8 h. Nonglycosylated GM2AP was obtained by labeling of hEKs in the simultaneous presence of 10 mM NH4Cl and 5 µg/ml tunicamycin. The secretions were dialyzed overnight against two 500-ml changes of MCDB 153 medium base in dialysis tubing with a 10-kDa cutoff to remove the inhibitors and were added to three 60-mm dishes of unlabeled cells in a total volume of 2 ml/dish. Endocytosis was allowed to proceed for 24 h. Afterward, the cells were extracted and immunoprecipitated as described below. Of the endocytosis media, 200 µl were filled up to 700 µl with cell extraction buffer (see below) and immunoprecipitated.

Preparation of Cell Extracts and Immunoprecipitation

After the pulse-chase experiment, the cells were washed twice with ice-cold phosphate-buffered saline and scraped off with 0.7 ml of ice-cold lysis buffer (phosphate-buffered saline containing 5 mM EDTA, 0.5% bovine serum albumin, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each leupeptin and pepstatin A). After the removal of insoluble matter by a 20-min centrifugation at 17,500 × g and 4 °C, 10 µl of the extracts were saved for acid precipitation. Immunoprecipitation was carried out as described (24), except that 3.5 µl of goat anti-GM2AP serum/ml of sample and 10 µl of protein G-Sepharose/µl of serum were used. Finally, the immune complexes were solubilized by boiling in 0.5% SDS/1% beta -mercaptoethanol.

Deglycosylation of the precipitates with endo H or PNGase F was carried out according to the manufacturer's instructions. The samples were subjected to reducing SDS gel electrophoresis in the Tris/Tricine buffer system of Schägger and von Jagow (25) using 12.5% polyacrylamide in the separating gel. Afterward, the gels were soaked in Amplify (Amersham), dried, exposed overnight to a Fuji BAS 1000 imaging system screen, and recorded with a Fuji BAS 1000 system. For a permanent record, the gels were exposed to Kodak BioMax MR x-ray film.

Magnetic Isolation of Lysosomes

The preparation of ferromagnetic dextran and the magnetic isolation of lysosomes were carried out essentially as described by Rodriguez-Paris et al. (26) with the following modifications.3 The cells were loaded with the iron/dextran probe for 24 h and chased for at least 10 h. They were harvested by trypsinization and lysed in homogenization buffer (27) supplied with 0.5 mM 4-aminoethylbenzylsulfonyl fluoride (Calbiochem) and 1 µg/ml each leupeptin and pepstatin A by 20 passages through a 24-gauge needle. Nuclei and insoluble components were removed by centrifugation (750 × g, 5 min, 4 °C). The supernatant was passed over a steel wool column placed in a magnetic cell-sorting device (Miltenyi Biotec, Bergisch-Gladbach, Germany). The flow-through was collected. After washing with homogenization buffer (5 × 1 ml), the column was removed from the magnet for elution of lysosomes. Nondestructive elution was achieved with 5 × 1 ml of homogenization buffer. Destructive elution was performed with 5 × 1 ml of water. Thereafter, each fraction was assayed for the following marker enzymes as described (28): alkaline phosphatase (plasma membrane), lactate dehydrogenase (cytoplasm), NADPH-dependent cytochrome-C reductase (endoplasmic reticulum), galactosyl-transferase (Golgi apparatus), and succinate dehydrogenase (mitochondria). beta -Hexosaminidase (lysosomes) was assayed as described (29) using (4-nitrophenyl)-beta -D-N-acetylglucosaminide as a substrate. For hEKs, it was found that 60-80% of the preloaded lysosomes were lost into the 750 × g pellet. However, losses of 40-60% were also observed with unlabeled cells. More than 90% of total protein and all nonlysosomal marker enzyme activities were recovered from the flow-through. Only 6 ± 2% of total hexosaminidase activity could be recovered from the nondestructive eluate and were contaminated by mitochondria and some plasma membrane, but sufficiently enriched lysosomal material was eluted destructively. 36 ± 10% of hexosaminidase was found in this fraction, being 65 ± 9-fold enriched (mean of six experiments in all cases) and being contaminated only by trace amounts of mitochondrial and plasma membrane marker activities.


RESULTS

Biosynthesis and Processing of GM2AP in hEKs

hEKs were pulse-labeled with [35S]cysteine for 1 h and chased for 2, 6, 24, and 48 h. GM2AP was immunoprecipitated from the media (Fig. 1A, left panel) and from cell extracts (Fig. 1A, right panel). GM2AP was not yet detectable in the medium after the pulse period, whereas an intense single band of 22 kDa was precipitated from the cell extract. After a 2-h chase, an additional band of 24 kDa appeared in the medium as well as in the cells. In the medium, the intensity of this band increased within the next 10 h but then remained constant during a chase time of 48 h. This secreted form of GM2AP was accompanied by at least three additional weaker bands of 25, 26, and 27 kDa and by traces of the intracellular 22-kDa form and an 18-kDa form. In the cells, however, the initial band doublet of 22 and 24 kDa was replaced during 24 h by a single, broad band centered around 20 kDa after having passed through several intermediate stages of less clearly defined molecular mass. This form persisted for at least 48 h without being further processed.


Fig. 1. Processing of GM2AP in hEKs. hEKs were pulse-labeled with [35S]cysteine for 1 h and chased for the times indicated. Media and cell lysates were immunoprecipitated for GM2AP, and the precipitates were separated by SDS-PAGE. A, glycosylated GM2AP; B, GM2AP after glycosidase treatment. The apparent molecular masses and glycosylation states are indicated at the right.
[View Larger Version of this Image (62K GIF file)]


Removal of the GM2AP N-glycan by endo H or PNGase F (Fig. 1B) revealed that most of the apparent molecular mass difference between individual GM2AP forms was due to carbohydrate processing events. The early 22-kDa band and the 24-27-kDa forms shared the same 18-kDa peptide backbone, but the 22-kDa GM2AP bore an endo H-sensitive carbohydrate, whereas the N-glycan of the 24-27-kDa GM2AP was endo H-resistant. Experiments with more closely spaced chase times (not shown) demonstrated that about 70% of the 22-kDa GM2AP was converted to the 24-27-kDa forms during the first 6 h of chase. At 6 h of chase, conversion of the 18-kDa peptide to a 17-kDa product was already detectable, indicating the onset of proteolytic processing on the peptide chain of GM2AP. Processing was complete after 24 h, and no endo H-sensitive glycoforms could be detected at this time any more.

For comparison, cathepsin L was subsequently immunoprecipitated from the cells and media (not shown). It was chosen as a representative of soluble lysosomal enzymes not involved into glycosphingolipid metabolism and because it has a unique N-glycosylation site (30), just like GM2AP. After 24 h of chase, a 42-kDa precursor was found in the medium, and two mature forms of 29 and 24.5 kDa (31) were detected intracellularly. The oligosaccharide side chains of all forms were entirely endo H-sensitive.

Fig. 2 summarizes the results for GM2AP schematically. It anticipates some of the observations given in detail later, but it is intended as a guide to the large number of GM2AP forms and their intracellular localization. In hEKs, GM2AP is synthesized as a precursor with a backbone size of 18 kDa after translocation into the ER and removal of the signal peptide. The precursor is cotranslationally glycosylated at its unique N-glycosylation site, yielding a 22-kDa protein with a high mannose type carbohydrate chain (precursor, high mannose (PHM)). Passing through the Golgi apparatus, a significant amount of this early precursor is converted to an endo H-resistant glycoform of 24 kDa, which most likely bears a complex type oligosaccharide (precursor, complex (PC)) and is partially secreted. Glycoforms of 25-27 kDa are generated from a minor fraction of the 24-kDa precursor immediately before its secretion. They presumably bear multiantennary glycan structures (precursor, multiantennary (PMA)), and only trace amounts of them are found inside the cell. A small portion (<= 5%) of the secretions consists of 22-kDa GM2AP. The secretion ceases between 6 and 12 h after synthesis, when about one-third of the total amount of precursor present after a 1-h pulse is found outside the cell. Simultaneously, late processing events occur on the protein backbone as well as on the oligosaccharide of the intracellular forms, suggesting that the bulk of GM2AP is then entering the lysosome. The intermediates of this late processing cover the entire molecular mass range between 20 and 24 kDa. After 24 h, processing has yielded a 20-kDa mature form (M) with a 17-kDa protein backbone. Its carbohydrate side chain is completely endo H-resistant and probably heavily trimmed, since the mass shift between the precursor glycoforms and M is 2 or 4 kDa, but proteolytic processing accounts for only 1 kDa of this apparent loss (compare with Fig. 1).


Fig. 2. Schematic representation of GM2AP processing in hEKs. The localization of individual glyco and protein forms of GM2AP during the course of transport and processing as determined by metabolic labeling and immunoprecipitation is shown. Thick arrows, major pathways; thin arrows, minor pathways. Approximate transit or conversion times are indicated for each step. Blocking agents are given for transitions that could be inhibited. BafA1, bafilomycin A1.
[View Larger Version of this Image (31K GIF file)]


The half-life of mature GM2AP must be well beyond the chosen chase times, since no significant decrease in overall signal intensity was observed even after 72 h of chase (not shown). Varying amounts (2-4%) of the total secreted material consisted of 18-kDa dP, suggesting that a part of GM2AP had escaped N-glycosylation intracellularly, since a trace of dP was also observed within the cells.

Carbohydrate Phosphorylation of GM2AP

hEKs were labeled with [33P]phosphate to assess which glycoform of GM2AP bears phosphomannosyl residues (Fig. 3). In the media, only a faint band of PHM appeared after the pulse period of 6 h, the label of which could completely be removed by endo H. In the cells, a single, broader band of 22-23 kDa was observed. In this case, 90% of the label was removed by endo H and the remainder by PNGase F, demonstrating that the carbohydrate rather than the backbone of GM2AP is phosphorylated, and that this marker resides predominantly on PHM. Since the labeling effectivity seemed to be rather low, we excluded the possibility of an inefficient label incorporation by sequential immunoprecipitation of cathepsins L and D and hexosaminidase beta -chain from the same samples. In all cases, the precursors and mature forms were easily detectable, exemplified for cathepsin L in Fig. 3, right panel. The phosphate-labeling efficiency of cathepsin L was 2.3-fold enhanced over that of GM2AP. Since the biosynthesis level of GM2AP was found to be 5.0 ± 0.6-fold enhanced over that of cathepsin L by [35S]cysteine labeling (data not shown), one may estimate that less than 10% of the GM2AP precursor is tagged with a M6P recognition marker.


Fig. 3. Carbohydrate phosphorylation of GM2AP and cathepsin L. hEKs were pulse-labeled with [33P]phosphate for 6 h and chased for the times indicated at the top. Media and cell lysates were immunoprecipitated for GM2AP and cathepsin L as indicated. The samples were separated by SDS-PAGE either directly or after glycosidase treatment as indicated (eH, endo H; PF, PNGase F). The positions of GM2AP forms are indicated at the left; those of cathepsin L forms are indicated at the right (P, precursor; M1, single chain mature form; M2, heavy chain of two chain mature form; Ref. 31). PHM was identified by its endo H sensitivity and by comparison to [35S]labeled GM2AP obtained from previous experiments (lanes removed).
[View Larger Version of this Image (49K GIF file)]


[35S]Sulfate labeling of hEK revealed that a tiny fraction of PC bears oligosaccharide-linked sulfate residues (not shown), but the signal intensity was close to the detection limit even after 14 h of pulse.

Processing of GM2AP in the Presence of Inhibitors of Early Stages of Intracellular Transport

Various inhibitors of intracellular transport were used to verify the precursor-product relationship for GM2AP and to assign individual GM2AP modifications to particular cellular compartments more closely. First, the GM2AP precursor was arrested in stages of early processing by a temperature shift and brefeldin A (BFA) treatment.

After a 1-h pulse at 37 °C, hEKs were chased at 14 °C for 24 h, which should trap early GM2AP forms by freezing vesicle transport between the ER and the Golgi apparatus (32) (Fig. 4, lanes 1-4). Only a trace of PC appeared in the medium (Fig. 4, lane 1). In the cells, more than 80% of the total signal still corresponded to PHM (Fig. 4, lanes 2-4), and no mature form appeared after this chase time. This is consistent with the view, that conversion of PHM to PC is a carbohydrate processing event caused by Golgi type modifications in the oligosaccharide chain of GM2AP. The small amounts of PC present at this point of time had not been processed to a mature form, which gave a clear indication that GM2AP matures in a compartment distal to the Golgi apparatus. Two additional hitherto unobserved products appeared after the temperature shift, one of 19 kDa and one of 16.5 kDa (Fig. 4, lane 2). Their origin remained obscure, and they disappeared after glycosidase treatment (Fig. 4, lanes 3 and 4), thus revealing that they did not represent M and dM. Release of the temperature block after 24 h fully restored the usual processing pattern (not shown).


Fig. 4. Processing of GM2AP in the presence of inhibitors of early stages of intracellular transport. hEKs were pulse labeled for 1 h with [35S]cysteine in the absence (lanes 1-4) or presence (lanes 5-14, 17, and 18) of 5 µg of BFA/ml of medium. They were chased for the times indicated, either at 14 °C (lanes 1-4) or in the presence of BFA (lanes 5-14, 17, and 18). For lanes 7, 13, and 14, BFA was removed afterward, and the cells were chased for another 24 h in the absence of the drug. The media (m) and cell lysates (c) were immunoprecipitated for GM2AP or cathepsin L (cath. L) as indicated, and the precipitates were separated by SDS-PAGE either directly or after glycosidase treatment (eH, endo H; PF, PNGase F). For lanes 15 and 16, the cells were pulse-labeled with [33P]phosphate for 6 h and chased for 24 h in the presence of BFA. The positions of GM2AP forms are indicated at the left; those of cathepsin L forms are indicated at the right. *, unknown contaminant precipitated with cathepsin L. Note that two GM2AP bands of undefined origin appear close to the expected positions of M and dM in lane 2, which disappear on glycosidase treatment (compare lanes 3 and 4).
[View Larger Version of this Image (38K GIF file)]


An unexpected result was obtained when the cells were exposed to BFA (5 µg/ml), a fungal macrolide antibiotic, which leads to an immediate vesiculation of the Golgi apparatus. It blocks anterograde vesicular transport from the ER but does not interfere with retrograde membrane flow between Golgi and the ER (33). In this respect, BFA would have been predicted to exert essentially the same effects on GM2AP transport and maturation as the 14 °C shift. Fig. 4, lanes 5-14, shows that this was not quite the case. If the cells were treated with BFA during the starvation period, a 1-h pulse, and a 6-h chase, the secretion of GM2AP was completely prevented (Fig. 4, lane 5). Within the cells, however, a band triplet of 22, 23, and 24 kDa was found (Fig. 4, lane 8) instead of the expected PHM. These forms proved to be largely endo H-resistant (Fig. 4, lane 9). After 24 h of chase in the presence of BFA, the triplet had been converted to PC (Fig. 4, lanes 10 and 11) with the usual dP backbone (Fig. 4, lane 12). This result also supported the view that the proteolytic processing events leading to the generation of the 17-kDa mature peptide must take place in a post-Golgi compartment, presumably in endosomes and/or lysosomes. No alteration of the N-glycan was observed for cathepsin L, the precursor still being in its high mannose form even after 24 h of chase in the presence of BFA (Fig. 4, lanes 17 and 18).

To assess whether the modified glycosylation pattern had any effect on GM2AP maturation, the cells were chased in the presence of BFA for 24 h and for another 24 h in the absence of the drug. In this case, secretion was reconstituted but rose to 50% (Fig. 4, lane 7). In the cells, PC was processed to mature GM2AP (Fig. 4, lanes 13 and 14), indicating that the complex glycoform of the GM2AP precursor is also competent for lysosomal transport. Checking for the phosphorylation state of the BFA-induced glycoforms, we treated hEKs with BFA during a 6-h pulse with [33P]phosphate and a 24-h chase. Only PHM was recovered from the cells (Fig. 4, lanes 15 and 16). Remarkably, PHM had completely escaped detection by [35S]cysteine labeling under these conditions (compare with Fig. 4, lanes 10 and 11), again arguing for the view that only a minor subfraction of GM2AP bears phosphomannosyl residues.

Processing of GM2AP in the Presence of Inhibitors of Late Stages of Intracellular Transport and Processing

Late stages of GM2AP transport and processing were disturbed by treating hEKs with ammonium chloride or protease inhibitors. Ammonium chloride induces hypersecretion of lysosomal enzyme precursors by increasing the intraorganellar pH of acidic compartments (34). The elevated pH probably prevents dissociation of the M6P recognition markers from M6P receptors. The occupied receptors are depleted from the Golgi apparatus, whereby newly synthesized ligands are diverted to the secretory pathway (35, 36). Leupeptin was chosen as an inhibitor of thiol proteases, and pepstatin A was chosen as an inhibitor of aspartyl proteases (37).

Maturation of GM2AP precursor was suppressed by ammonium chloride treatment of hEKs (Fig. 5, left panel). High amounts of PC were secreted into the chase media. After 6 h of chase, both PHM and PC were immunoprecipitated from the cells. Only 10% of total GM2AP remained in the cells after 24 h of chase, which were in the PC form and not processed to mature GM2AP. Similar results were obtained (not shown) when hEKs were exposed to bafilomycin A1, an inhibitor of the vesicular proton pump (38), or to monensin, a Na+ and K+ ionophore, which disturbs transport at a late Golgi stage (39).


Fig. 5. Processing of GM2AP in the presence of NH4Cl. hEKs were pulse labeled for 1 h with [35S]cysteine in the presence of 10 mM NH4Cl and chased for the times indicated. Media and cell lysates were immunoprecipitated for GM2AP and cathepsin L as indicated. The positions of individual GM2AP forms are indicated at the left; those of cathepsin L forms are indicated at the right. Band designations are as for Figs. 1 (GM2AP) and 3 (cathepsin L). *, unknown contaminant precipitating with cathepsin L.
[View Larger Version of this Image (41K GIF file)]


In contrast, 60% of the cathepsin L precursor had been secreted after 24 h of chase in the presence of NH4Cl, but the remaining 40% had been converted to the 29-kDa form intracellularly (Fig. 5, right panel). The 24.5-kDa form was not generated, possibly because the autoprocessing of cathepsin L (40) is inhibited at elevated pH.

Apparently, the proper targeting of GM2AP in hEKs is strongly dependent on acidification of the compartments responsible for late sorting steps, whereas the targeting of cathepsin L is less restricted in this respect.

Treatment of hEKs with leupeptin (0.1 mM) completely prevented maturation of the GM2AP peptide backbone (not shown). Instead of 20-kDa mature GM2AP, a 21-kDa protein was precipitated from the cells after 24 h, from which dP (18 kDa) was liberated by PNGase F treatment. Thus, a thiol protease is involved in the maturation of the GM2AP precursor, and the late processing of its N-glycan may occur independently from the proteolytic step. Treating the cells with pepstatin A (0.1 mM) had no effect on GM2AP maturation.

Transport of Nonglycosylated GM2AP

The significance of the carbohydrate for lysosomal targeting of GM2AP was probed by desoxymanno-nojirimycin and swainsonine treatment, respectively (not shown). These compounds are potent inhibitors of Golgi mannosidases prematurely terminating the trimming of N-glycans at high mannose stages (reviewed in Ref. 41). The maturation of GM2AP was not affected by these drugs, but both inhibitors led to the generation of entirely endo H-sensitive glycoforms, including even secreted and mature GM2AP. In conjunction with the results of the BFA treatment presented in Fig. 4, it therefore seemed questionable whether the carbohydrate of GM2AP has any significance for its lysosomal delivery at all. Consequently, we suppressed the cotranslational attachment of its N-glycan by tunicamycin treatment of hEKs. Tunicamycin interferes with the biosynthesis of the dolicholpyrophosphate precursor of N-linked carbohydrate side chains, thereby globally preventing N-glycosylation (41, 42).

hEKs were treated with tunicamycin (5 µg/ml) during the starvation period and a 1-h pulse with [35S]cysteine. They were chased for 0, 6, and 24 h in the absence of the drug and immunoprecipitated for GM2AP (Fig. 6). Qualitatively, the same GM2AP band pattern was obtained as for deglycosylated samples from pulse-chase experiments conducted without tunicamycin (compare with Fig. 1B). dP was precipitated from the media, the intensity of which increased over 24 h of chase. In the cells, dP appeared after the pulse, showing first signs of proteolytic processing after 6 h of chase and having been completely converted to dM after 24 h. Tunicamycin treatment seemed to have no adverse effect on GM2AP synthesis and stability. Overall protein synthesis dropped to 70% as judged by trichloroacetic acid precipitation of the lysates, but the immunoprecipitable proportion of GM2AP remained constant under these conditions. The combined intensity of dP and dM did not significantly decrease over the chase period. The kinetics of GM2AP transport also seemed to be unaffected compared with the normal situation. However, two additional bands (16 and 14 kDa) appeared, which had not been observed in the absence of the inhibitor and which were not subjected to further proteolytic processing. Again, the secretion of the GM2AP precursor rose to 50% instead of the normal 30%, which had also been observed after release of the BFA block.


Fig. 6. Processing of nonglycosylated GM2AP and cathepsin L. hEKs were pulse-labeled for 1 h with [35S]cysteine in the presence of 5 µg of tunicamycin/ml of medium and chased in the absence of the drug for the times indicated. The media (m) and cell lysates (c) were immunoprecipitated for GM2AP or cathepsin L as indicated. The positions of individual protein forms are given at the left for GM2AP and at the right for cathepsin L. *, GM2AP bands of undefined origin.
[View Larger Version of this Image (35K GIF file)]


Quite another situation was met with cathepsin L (Fig. 6, right four lanes). Here, a nonglycosylated precursor of 38 kDa was synthesized and completely excreted into the medium. Similar results were obtained for cathepsin D (not shown).

This experiment indicated that an alternative pathway for lysosomal targeting exists in hEKs, which allows effective delivery of GM2AP to the lysosome even when no N-glycan is attached to its protein backbone. This mechanism obviously does not operate on the precursors of cathepsins L and D.

In an attempt to determine whether the transport of GM2AP or cathepsin L is a membrane-associated process, we tried to recover their precursors from the cellular scaffold of hEKs differentially permeabilized with saponin according to the method of Rijnboutt et al. (19). However, GM2AP as well as cathepsin L could only be detected in the saponin supernatant, not in the membrane pellet (not shown).

Lysosomal Localization of Mature GM2AP by Magnetic Isolation of hEK Lysosomes

To prove the lysosomal localization of processed GM2AP, hEK lysosomes were isolated by magnetic fractionation.

For cold experiments, hEKs were preloaded for 24 h with a probe of colloidal iron coupled to dextran, which was ingested by nonspecific endocytosis. After a chase of at least 10 h, the cells were lysed, a 750 × g supernatant was subjected to magnetic separation of lysosomes, and the fractions were assayed for the distribution of subcellular marker enzyme activities. The bulk of activity of the marker enzymes for cytoplasm, plasma membrane, mitochondrial membrane, ER, and Golgi was recovered from the flow-through. About 54% of total hexosaminidase activity was also eluted here, most probably having been liberated from disrupted lysosomes. Hexosaminidase was enriched 65 ± 9-fold in the fractions eluted destructively and depleted 2.5 ± 0.5-fold from the flow-through.

For the localization of nonglycosylated GM2AP, hEKs were pulse-labeled with [35S]cysteine for 5 h in the presence or absence of tunicamycin. They were chased for 14 h in the presence of the magnetic probe and for another 10 h in its absence. Fig. 7 shows that mature GM2AP could be immunoprecipitated from the flow-through and, more significantly, from the lysosomal fraction of the magnetic separation, regardless of whether it was glycosylated or not. GM2AP was 2-fold depleted from the flow-through and about 45-fold enriched in the destructive eluates from these separations, which essentially parallels the hexosaminidase enrichment given above.


Fig. 7. Lysosomal localization of GM2AP. hEKs were pulse-labeled with [35S]cysteine for 5 h in the presence or absence of 5 µg of tunicamycin/ml of medium as indicated. The cysteine label was chased by loading the cells with s.c. in complete medium for 14 h. Afterward both the magnetic probe and the cysteine label were chased for another 10 h. The cells were subjected to magnetic separation of lysosomes as described under "Experimental Procedures." The combined flow-through and wash fractions (flow-through) as well as the destructively eluted fractions (eluate) were immunoprecipitated for GM2AP, and the samples were separated by SDS-PAGE. The positions of individual GM2AP forms are given at the right, with designations according to Fig. 1. *, sample carryover from neighboring lanes, which have been removed. GM2AP distribution and enrichment are indicated below the panel and were calculated from the immunoprecipitable proportion of GM2AP relative to total protein.
[View Larger Version of this Image (46K GIF file)]


In this manner, we showed that in hEKs, GM2AP is properly targeted to the lysosome even if no N-glycan is cotranslationally attached to its unique N-glycosylation site.

Endocytosis of GM2AP

We examined whether the carbohydrate would be of any relevance for the uptake of exogenously added GM2AP by offering the [35S]cysteine-labeled secretions of ammonium chloride-treated hEK to unlabeled cells for a 24-h period in the presence of M6P or glucose-6-phosphate (10 mM each) or yeast mannan or asialo-orosomucoid (1 mg/ml each) or a combination of M6P, mannan, and asialo-orosomucoid at the above concentrations, respectively. These inhibitors were chosen to interfere with endocytosis by the receptors for M6P, exposed mannoses, or asialoglycoproteins (43). The cells and 10% of their media were sequentially immunoprecipitated for GM2AP, cathepsin L, and hexosaminidase beta -chain (Fig. 8).


Fig. 8. Endocytosis of GM2AP and other lysosomal proteins in hEKs. hEKs were labeled with [35S]cysteine for 8 h in the presence of 10 mM NH4Cl. The secretions were collected, dialyzed against fresh medium, and added for 24 h to unlabeled cells in the presence of no inhibitor, 10 mM M6P, 10 mM glucose-6-phosphate (G6P), 1 mg/ml yeast mannan, 1 mg/ml asialo-orosomucoid (ASOM), or a combination of M6P and the latter two at the above concentrations, respectively (indicated at the bottom). Afterward, the cell lysate and 10% of the medium were immunoprecipitated for GM2AP (left panel). The untreated controls and M6P-treated cells were also immunoprecipitated for cathepsin L and hexosaminidase beta -chain (right panel), and the samples were separated by SDS-PAGE. The positions of individual protein forms are indicated on both sides of each panel. Hexosaminidase bands were assigned according to Ref. 61. alpha , traces of hexosaminidase alpha -chain coprecipitating with beta -chains from heterodimeric hexosaminidase A; beta b(2), singly glycosylated form of beta b peptide (62). s, molecular mass standard (14.3, 21.3, and 30 kDa are visible in the left panel; 14.3, 21.3, 30, 46, and 69 kDa are visible in the right panel).
[View Larger Version of this Image (45K GIF file)]


Three additional GM2AP bands appeared in the medium besides PC and PMA, which were assigned to PHM, dP, and an unknown component according to their molecular masses. The endocytosis of GM2AP was not significantly affected by any of the inhibitors (Fig. 8, left panel). Under M6P, glucose-6-phosphate, mannan, and asialo-orosomucoid alone, the uptake was 95-120% of the untreated control. It dropped to 75% in the presence of the inhibitor mixture, which we believe to be a nonspecific effect due to overloading of the serum-free medium with unusual additives, since none of the inhibitors had an adverse effect when applied alone. The endocytosis of cathepsin L was only slightly affected by M6P, whereas the uptake of hexosaminidase beta -chain was almost completely inhibited in the presence of M6P (Fig. 8, right panel). The relative molar concentrations offered in the media were calculated to be 4.4:1:1.1 for GM2AP, cathepsin L, and hexosaminidase beta -chain, respectively. After 24 h of endocytosis in the absence of any inhibitor, 4.1 ± 0.5% of the added material were recovered from the cells for GM2AP, 5.0 ± 0.5% for cathepsin L, and 12.5 ± 1.5% for hexosaminidase beta -chain, respectively (mean of two experiments). These variations may reflect either the individual rates of uptake and/or different rates of degradation.

A second endocytosis experiment was conducted as above, except that the secretions were collected from cells that had been labeled in the additional presence of tunicamycin and that no inhibitor was present during the endocytosis phase (not shown). Under these conditions, GM2AP was the only one of the four referenced antigens (GM2AP, cathepsins L and D, and hexosaminidase beta -chain) that was endocytosed and processed effectively. Surprisingly, even 8.5 ± 1% (mean of two experiments) of the offered GM2AP was recovered from the cells after 24 h in this case.

Both findings indicate that endocytosis of GM2AP is largely independent of the known N-glycan-linked signals for receptor-mediated endocytosis in hEKs.


DISCUSSION

In this article, we have clearly established that in hEKs, GM2AP is synthesized as a 22-kDa precursor (PHM) bearing a single high mannose N-linked oligosaccharide chain on a peptide backbone of 18 kDa. In the Golgi apparatus, at least 70% of PHM is converted to 24-kDa PC by remodeling of the N-glycan to a complex type oligosaccharide. About one-third of the GM2AP precursor is secreted, and more than 90% of these secretory forms consist of PC. The intracellular remainder is segregated from the secretory pathway and processed in a post-Golgi compartment to yield a 20-kDa mature form (M), which bears an endo H-resistant N-glycan on a 17-kDa peptide (summarized in Fig. 2).

Thus, the early and late processing of GM2AP shows many features typical of soluble lysosomal enzymes (reviewed in Ref. 44). It is synthesized as a precursor protein, the N-glycan of which receives Golgi type modifications. It is partially secreted into the medium, and it becomes subject to proteolytic and glycolytic processing events in acidic compartments after segregation. The molecular mass of the major secreted form (PC, 24 kDa) was in agreement with earlier findings (10). Regarding the increased half-width of the mature GM2AP band, one may assume that M is microheterogeneous in its N-glycan and/or its protein backbone. It should be noted in this context that two protein species were also present in M purified from human kidney: one major form with Phe34 and a minor one with Ser32 as the amino terminus (45). The molecular mass found for dM (17 kDa by SDS-PAGE) is close to its calculated molecular mass from cDNA and protein sequencing data (17,369 Da starting from Phe34). Since dP has an apparent molecular mass of 18 kDa, it is very likely that the signal peptide of GM2AP is removed on entry into the ER. The algorithm of von Heijne (46) predicts signal peptidase cleavage sites at Gln22 or His24, leading to precursors of 18,561 or 18,362 Da, respectively.

Very recently, a report by Wu et al. (47) proposed that an alternatively spliced form of GM2AP (Met1-Glu142 plus Val-Ser-Thr, termed GM2AP(A) below) might exist in human placenta and fibroblasts, which in vitro has an activating effect on clostridial sialidase but not on hexosaminidase. Taking into account the expected decrease in molecular mass of such a splice variant (-5 kDa for all forms), we rescreened our fluorographs for the presence of GM2AP(A). At least for hEKs, we were not able to detect GM2AP-related products in the relevant molecular mass window. Shortened GM2AP forms appeared only when inhibitors of early transport (see Fig. 4, lane 2) or tunicamycin (see Fig. 6) were used. However, they were not processed in the same manner as full-size GM2AP, and we assume that they represent prematurely and aberrantly processed forms arising from the presence of the inhibitors.

PC first appears in the medium 60-90 min after synthesis, a value that is typical for other lysosomal enzymes too (44). In hEKs, however, its secretion does not cease before 12 h after synthesis, and its final maturation takes more than 12 h. Similar kinetics were observed for cathepsin L. It seems that segregation and transport of at least these two lysosomal proteins is somewhat delayed between late Golgi and lysosomal compartments. To our knowledge, this is the first study dealing with the processing of lysosomal proteins in hEKs. Since the maturation of lysosomal protein precursors occurs in ranges from minutes to days depending on cell type and species (44), we attribute these slowed-down kinetics to a yet uncharacterized effect specific to hEK.

Furthermore, we have shown that GM2AP is targeted to the lysosomes by a M6P-independent pathway in hEKs. After Klima et al. (12) had found that recombinant, nonglycosylated GM2AP is effectively endocytosed by human fibroblasts, the question arose whether its intracellular transport would also be at least partially independent of M6P or other carbohydrate-linked signals. Several different lines of evidence demonstrated that such a pathway must exist in hEKs.

First, we have shown that only a minor fraction of GM2AP, possibly 10%, bears phosphomannosyl residues, and that this tag is confined to PHM exclusively. Since at least 70% of PHMs are converted to PCs and since 70% of GM2AP precursors are retained intracellularly, a significant proportion of M should be derived from PC. The endo H resistance of M also suggests that it may be derived mainly from a precursor with complex type oligosaccharides. It has to be kept in mind, however, that extensive trimming of mannose-rich N-glycans may abolish recognition by endo H (44). In addition, we could trap PC exclusively by BFA treatment and observed that it is correctly processed to M on detoxification of the cells (Fig. 4, lanes 13 and 14). Under BFA, the oligosaccharide of PHM was remodeled almost instantaneously by the action of Golgi glycosidases and glycosyltransferases, which retain at least part of their activity in the fused ER-Golgi compartment (49). It may well be that the underphosphorylated N-glycan of GM2AP is a preferred substrate for such modifications, since M6P serves as a protecting group against Golgi glycosidase trimming (44, 48, 50). Indeed, the efficiently phosphorylated carbohydrate of cathepsin L was under no circumstances converted to a complex form in hEKs.

Second, only PC is hypersecreted on NH4Cl treatment. For enzymes sorted via M6P receptors, NH4Cl interferes with sorting in a post-Golgi compartment, and the secretions are highly enriched in phosphorylated precursor glycoforms (35). Therefore, it is likely that PC passes a similar base-sensitive compartment, and the high selectivity by which NH4Cl induces its hypersecretion and not that of PHM suggests that it is mainly PC that moves through this compartment. PC would then be the dominant precursor to M, implying that the M6P receptor system possibly plays only a minor role in the segregation of GM2AP, since PC does not bear phosphomannosyl residues.

Finally, and most strikingly, we found that GM2AP was correctly targeted at an apparently normal rate even when its glycosylation had been suppressed by tunicamycin treatment (Figs. 6 and 7), suggesting that the lysosomal targeting signal resides on its protein backbone rather than on its carbohydrate. Interestingly, the secretion of the GM2AP precursor rose from 30 to 50% under tunicamycin. This difference is close to the 10% value estimated to be the mannose-6-phosphorylated fraction of GM2AP. Usually, tunicamycin treatment strongly enhances the secretion of lysosomal enzymes transported in response to the M6P system (51-53), which then are shed from the cells as unprocessed precursors (52, 53). In this respect, the nonglycosylated precursors of cathepsins L and D behaved as expected.

Thus, the intracellular transport of GM2AP adds to an increasing number of reports about correct targeting of lysosomal enzymes independent of phosphomannosyl residues. Such phenomena have been described for cathepsin D in HepG2 cells (14, 19), in breast cancer cells (15), and in ICD lymphoblasts (16), as well as for beta -aspartylglucosaminidase (17) and alpha -glucosidase (18) in transfected COS1 cells. In the cases of cathepsin D (14, 15, 19, 54), alpha -glucosidase (18), glucocerebrosidase and sulfatide activator protein (19), and cathepsin L (55), a M6P-independent membrane association of the precursors was observed. We could not demonstrate a membrane association of the GM2AP precursor by saponin permeabilization, but we cannot exclude a weak interaction, which may be disrupted by this treatment. For alpha -glucosidase, the membrane association is mediated by an uncleaved signal peptide (18). However, our data indicate that the signal peptide of GM2AP is instantaneously removed on entry into the ER.

Since the function of GM2AP is to serve as a lipid-binding protein (1), and since GM2AP is able to transfer gangliosides from donor to acceptor liposomes in vitro (56), an intriguing option would be that it is transported and segregated in direct association to the lipid bilayer. Such an interaction has also been proposed for glucocerebrosidase, which does not bear M6P residues (57) and is possibly bound to acidic phospholipids (58).

Comparing the transport of GM2AP and cathepsin L in hEKs, it seems that two different targeting mechanisms operate on these proteins. The efficient segregation of cathepsin L is crucially dependent on the presence of an N-glycan, which is not the case for GM2AP. On the other hand, acidification of the transporting machinery is essential for the delivery of GM2AP, whereas it is less substantial to the targeting of cathepsin L. In this respect, each of both proteins displays one feature typical of M6P-dependent and one typical of M6P-independent transport. Obviously, the unknown recognition system responsible for GM2AP segregation shows a similar pH dependence as the M6P-receptor system. The transport of cathepsin L might proceed in a manner analogous to myelocytes and macrophages, in which base-sensitive and base-insensitive packaging mechanisms are known to exist in parallel (reviewed in Ref. 44).

The endocytosis of GM2AP is also independent of the known signals for carbohydrate-mediated internalization (Fig. 8). We had expected that M6P would not affect its uptake, since more than 90% of the offered secretions are made up of PC and the PMA, which are not phosphorylated. However, its endocytosis remained equally unaffected by inhibitors of mannose and asialoglycoprotein receptors. Nonglycosylated GM2AP is also internalized, as had already been shown earlier for human fibroblasts (12), but we were surprised to find that its uptake seemed almost twice as effective as for the glycosylated form. A similar observation has been made for the sphingolipid activator protein precursor in fibroblasts (24), and it has been suggested that the carbohydrate might partially mask an epitope involved in recognition between this protein and a yet unknown receptor system on the cell surface. It may again be speculated that GM2AP reaches the interior of the cell by binding to lipid components of the cell surface.

In the granular layer of the epidermis, keratinocytes start to form acidic lamellar granules, which contain precursors of specialized stratum corneum lipids, lysosomal hydrolases, and a vesicular ATP-dependent proton pump (reviewed in Refs. 59 and 60). The contents of these storage bodies is eventually shed into the uppermost, cornified layer of the epithelium. The present work was performed with basal keratinocytes, in which the basal characteristics were maintained by culture at low calcium concentrations (0.1 mM), suppressing the onset of terminal differentiation. Further experiments, however, may address the question of whether GM2AP is also targeted to the lamellar granules in differentiated keratinocytes and whether this targeting involves the same lysosomal type delivery mechanism as observed in basal keratinocytes, or whether a separate routing strategy is used. Additionally, future work may reveal the nature of the mechanisms responsible for the M6P-independent intracellular targeting and uptake of GM2AP and whether both of these processes are mediated by the same or different targeting systems.


FOOTNOTES

*   This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to the memory of Dr. W. Wille (1946-1992).


   To whom correspondence should be addressed. Tel.: 49-228-73-53-46; Fax: 49-228-73-77-78; E-mail: sandhoff{at}uni-bonn.de.
1    The abbreviations used are: GM2 ganglioside, GalNAc beta 1 right-arrow 4Gal(3 left-arrow  2alpha NeuNAc)beta 1 right-arrow 4Glc beta 1 right-arrow 1 ceramide; GM2AP, GM2 activator protein; BFA, brefeldin A; endo H, beta -endo-N-acetylglucosaminidase H; ER, endoplasmic reticulum; hEK, human epidermal keratinocyte; beta -hexosaminidase, 2-acetamido-2-deoxy-beta -D-hexoside acetamidodeoxyhexohydrolase (EC 3.2.1.52); M6P, mannose-6-phosphate; PNGase F, peptide-N-glycanase F; PAGE, polyacrylamide gel electrophoresis; PHM, precursor, high mannose; PC, precursor, complex; PMA, precursor with multiantennary N-glycan; M, mature GM2AP; dP, deglycosylated GM2AP precursor peptide; dM, deglycosylated GM2AP mature peptide.
2    G. J. Glombitza, and K. Sandhoff, unpublished observations.
3    E. Becker, G. J. Glombitza, and K. Sandhoff, unpublished procedures.

Acknowledgments

We are highly indebted to D. Beichelt and A. Issberner, who continuously provided us with excellent preparations of hEK. We thank the group of T. L. Steck for an introduction to magnetic organelle separation, M. Feldhoff, A. Raths, and J. Weisgerber for their technical assistance in cell culture, and the crew of Sander Photographie (Cologne, Germany), especially S. Erhard-Glombitza, for their expert assistance with the photographs. We gratefully acknowledge the critical comments of V. Gieselmann (Biochemisches Institut der Christian-Albrechts-Universität, Kiel, Germany), who carefully read this manuscript.


REFERENCES

  1. Sandhoff, K., Harzer, K., and Fürst, W. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds), Vol. II, pp. 2427-2441, McGraw Hill, New York
  2. Fürst, W., and Sandhoff, K. (1992) Biochim. Biophys. Acta 1126, 1-16 [Medline] [Order article via Infotrieve]
  3. Fürst, W., Machleidt, W., and Sandhoff, K. (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 [Medline] [Order article via Infotrieve]
  4. O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101 [Medline] [Order article via Infotrieve]
  5. Nakano, T., Sandhoff, K., Stümper, J., Christomanou, H., and Suzuki, K. (1989) J. Biochem. (Tokyo) 105, 152-154 [Abstract]
  6. Burg, J., Conzelmann, E., Sandhoff, K., Solomon, E., and Swallow, D. M. (1985) Ann. Hum. Genet. 49, 41-45 [Medline] [Order article via Infotrieve]
  7. Conzelmann, E., and Sandhoff, K. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3979-3983 [Abstract]
  8. Gravel, R. A., Clarke, J. T. R., Kaback, M. M., Mahuran, D., Sandhoff, K., and Suzuki, K. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds), Vol. II, pp. 2839-2879, McGraw Hill, New York
  9. Li, S. C., Wu, Y. Y., Sugiyama, E., Taki, T., Kasama, T., Casellato, R., Sonnino, S., and Li, Y. T. (1995) J. Biol. Chem. 270, 24246-24251 [Abstract/Free Full Text]
  10. Burg, J., Banerjee, A., and Sandhoff, K. (1985) Biol. Chem. Hoppe-Seyler 366, 887-891 [Medline] [Order article via Infotrieve]
  11. Kornfeld, S., and Mellman, I. (1989) Annu. Rev. Cell. Biol. 5, 483-525 [CrossRef]
  12. Klima, H., Klein, A., van Echten, G., Schwarzmann, G., Suzuki, K., and Sandhoff, K. (1993) Biochem J. 294, 227-230 [Medline] [Order article via Infotrieve]
  13. Waheed, A., Pohlmann, R., Hasilik, A., von Figura, K., van Elsen, A., and Leroy, J. G. (1982) Biochem. Biophys. Res. Commun. 105, 1052-1058 [Medline] [Order article via Infotrieve]
  14. Rijnboutt, S., Kal, A. J., Geuze, H., Aerts, H., and Strous, G. J. (1991) J. Biol. Chem. 266, 23586-23592 [Abstract/Free Full Text]
  15. Capony, F., Braulke, T., Rougeot, C., Roux, S., Montcourrier, P., and Rochefort, H. (1994) Exp. Cell Res. 215, 154-163 [CrossRef][Medline] [Order article via Infotrieve]
  16. Glickman, J. M., and Kornfeld, S. (1993) J. Cell Biol. 123, 99-108 [Abstract]
  17. Tikkanen, R., Enomaa, N., Riikonen, A., Ikonen, E., and Peltonen, L. (1995) DNA Cell. Biol. 14, 305-312 [Medline] [Order article via Infotrieve]
  18. Wisselaar, H. A., Kroos, M. A., Hermans, M. M. P., van Beeumen, J., and Reuser, A. (1993) J. Biol. Chem. 268, 2223-2231 [Abstract/Free Full Text]
  19. Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868 [Abstract/Free Full Text]
  20. Zhu, Y., and Conner, G. E. (1994) J. Biol. Chem. 269, 3846-3851 [Abstract/Free Full Text]
  21. Rheinwald, J. G., and Green, H. (1975) Cell 6, 331-344 [Medline] [Order article via Infotrieve]
  22. Pillai, S., Bikle, D. D., Hincenbergs, M., and Elias, P. M. (1988) J. Cell. Physiol. 134, 229-237 [Medline] [Order article via Infotrieve]
  23. Schröder, M., Schnabel, D., Hurwitz, R., Young, E., Suzuki, K., and Sandhoff, K. (1993) Hum. Genet. 92, 437-440 [Medline] [Order article via Infotrieve]
  24. Vielhaber, G., Hurwitz, R., and Sandhoff, K. (1997) J. Biol. Chem. 272, 32438-32446
  25. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  26. Rodriguez-Paris, J. M., Nolta, K. V., and Steck, T. L. (1993) J. Biol. Chem. 268, 9110-9116 [Abstract/Free Full Text]
  27. Padh, H., Lavasa, M., and Steck, T. L. (1989) J. Cell Biol. 108, 865-874 [Abstract]
  28. Graham, J. M. (1993) in Biomembrane Protocols: Methods in Molecular Biology (Graham, J. M., and Higgins, J. A., eds), pp. 1-18, Humana Press, Totowa, NJ
  29. Burg, J., Banerjee, A., Conzelmann, E., and Sandhoff, K. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 821-829 [Medline] [Order article via Infotrieve]
  30. Gal, S., and Gottesman, M. M. (1988) Biochem. J. 253, 303-306 [Medline] [Order article via Infotrieve]
  31. Erickson, A. H. (1989) J. Cell. Biochem. 40, 31-41 [Medline] [Order article via Infotrieve]
  32. Saraste, J., and Kuismanen, E. (1984) Cell 38, 535-549 [Medline] [Order article via Infotrieve]
  33. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080 [Medline] [Order article via Infotrieve]
  34. Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3327-3331 [Abstract]
  35. Gonzalez-Noriega, A., Grubb, J. H., Talkad, V., and Sly, W. S. (1982) J. Cell Biol. 95, 536-542 [Abstract/Free Full Text]
  36. Hasilik, A., and Neufeld, E. F. (1980) J. Biol. Chem. 255, 4937-4945 [Free Full Text]
  37. Aoyagi, T., and Umezawa, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D. B., and Shaw, E., eds), Vol. 2, pp. 429-439, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  38. Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., and Tashiro, Y. (1991) J. Biol. Chem. 266, 17707-17712 [Abstract/Free Full Text]
  39. Tartakoff, A. M. (1983) Cell 32, 1026-1028 [Medline] [Order article via Infotrieve]
  40. Salminen, A., and Gottesman, M. M. (1990) Biochem. J. 272, 39-44 [Medline] [Order article via Infotrieve]
  41. Fuhrmann, U., Bause, E., and Ploegh, H. (1985) Biochim. Biophys. Acta 825, 95-110 [Medline] [Order article via Infotrieve]
  42. Tkacz, J. S., and Lampen, O. (1975) Biochem. Biophys. Res. Commun. 65, 248-257 [Medline] [Order article via Infotrieve]
  43. Köster, A., von Figura, K., and Pohlmann, R. (1994) Eur. J. Biochem. 224, 685-689 [Abstract]
  44. Hasilik, A. (1992) Experientia (Basel) 48, 130-151 [Medline] [Order article via Infotrieve]
  45. Fürst, W., Schubert, J., Machleidt, W., Meyer, H. E., and Sandhoff, K. (1990) Eur. J. Biochem. 192, 709-714 [Abstract]
  46. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  47. Wu, Y. Y., Sonnino, S., Li, Y. T., and Li, S. C. (1996) J. Biol. Chem. 271, 10611-10615 [Abstract/Free Full Text]
  48. Goldberg, D., Gabel, C., and Kornfeld, S. (1984) in Lysosomes in Biology and Pathology (Dingle, J. T., Dean, R. T., and Sly, W. S., eds), pp. 45-62, Elsevier, Amsterdam
  49. Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S., and Klausner, R. D. (1989) Cell 56, 801-813 [Medline] [Order article via Infotrieve]
  50. Hasilik, A., and von Figura, K. (1981) Eur. J. Biochem. 121, 125-129 [Abstract]
  51. von Figura, K., Rey, M., Prinz, R., Voss, B., and Ullrich, K. (1979) Eur. J. Biochem. 101, 103-109 [Medline] [Order article via Infotrieve]
  52. Imort, M., Zühlsdorf, M., Feige, U., Hasilik, A., and von Figura, K. (1983) Biochem. J. 214, 671-678 [Medline] [Order article via Infotrieve]
  53. Rosenfeld, M. G., Kreibisch, G., Popov, D., Kato, K., and Sabatini, D. D. (1982) J. Cell Biol. 93, 135-143 [Abstract/Free Full Text]
  54. Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907 [Abstract/Free Full Text]
  55. McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445 [Abstract/Free Full Text]
  56. Conzelmann, E., Burg, J., Stephan, G., and Sandhoff, K. (1982) Eur. J. Biochem. 123, 455-464 [Abstract]
  57. Aerts, H. M. F. G., Schram, A. W., Strijland, A., van Weely, S., Jonsson, L. M. V., Tager, J. M., Sorell, S. H., Ginns, E. I., Barranger, J. A., and Murray, G. J. (1988) Biochim. Biophys. Acta 964, 303-308 [Medline] [Order article via Infotrieve]
  58. Imai, K. (1985) J. Biochem. (Tokyo) 98, 1405-1416 [Abstract]
  59. Watt, F. M. (1989) Curr. Opin. Cell Biol. 1, 1107-1115 [Medline] [Order article via Infotrieve]
  60. Fuchs, E. (1990) J. Cell Biol. 111, 2807-2814 [Medline] [Order article via Infotrieve]
  61. Mahuran, D. J., Neote, K., Klavins, M. H., Leung, A., and Gravel, R. A. (1988) J. Biol. Chem. 263, 4612-4618 [Abstract/Free Full Text]
  62. Sonderfeld-Fresko, S., and Proia, R. L. (1989) J. Biol. Chem. 264, 7692-7697 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.