(Received for publication, September 25, 1996, and in revised form, November 19, 1996)
From the 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
The processing, intracellular transport, and
endocytosis of the GM2 activator protein
(GM2AP), an essential cofactor of -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.
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 -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 -aspartylglucosaminidase (17) and
-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.
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
-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
1-acid glycoprotein,
human) was a kind gift of Dr. G. Schwarzmann (Institut fur
Organische Chemie und Biochemie, Universität Bonn, Bonn,
Germany).
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.
AntibodiesA 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--hexosaminidase
-chain was a kind gift of Dr.
R. Proia (NIDDK, National Institutes of Health, Bethesda, MD).
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 ExperimentsEpidermal 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 ImmunoprecipitationAfter
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% -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 LysosomesThe 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). -Hexosaminidase (lysosomes) was assayed as described (29) using
(4-nitrophenyl)-
-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.
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.
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).
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 GM2APhEKs 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 -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.
[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 TransportVarious 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).
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 ProcessingLate 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).
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 GM2APThe 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.
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 LysosomesTo 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.
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 GM2APWe 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 -chain (Fig. 8).
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 -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
-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
-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 -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.
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
-aspartylglucosaminidase (17) and
-glucosidase (18) in
transfected COS1 cells. In the cases of cathepsin D (14, 15, 19, 54),
-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
-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.
Dedicated to the memory of Dr. W. Wille (1946-1992).
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.