(Received for publication, February 10, 1997, and in revised form, March 15, 1997)
From the Glycobiology Institute, University of Oxford, South Parks
Road, OX1 3QU Oxford, United Kingdom and the Institute of
Biochemistry, Splaiul Independentei 296, 77700 Bucharest
17, Romania
Tyrosinase is the key enzyme in melanin
biosynthesis, catalyzing multiple steps in this pathway. The mature
glycoprotein is transported from the Golgi to the melanosome where
melanin biosynthesis occurs. In this study, we have investigated the
effects of inhibitors of N-glycan processing on the
synthesis, transport, and catalytic activity of tyrosinase. When B16
mouse melanoma cells were cultured in the presence of
N-butyldeoxynojirimycin, an inhibitor of the endoplasmic
reticulum-processing enzymes -glucosidases I and II, the
enzyme was synthesized and transported to the melanosome but almost
completely lacked catalytic activity. The cells contained only 2% of
the melanin found in untreated cells. Structural analysis of the
N-glycans from N-butyldeoxynojirimycin-treated
B16 cells demonstrated that three oligosaccharide structures
(Glc3Man7-9) predominated. Removal of
the glucose residues with
-glucosidases I and II failed to
restore enzymatic activity, suggesting that the glucosylated
N-glycans do not sterically interfere with the enzyme's
active sites. The mannosidase inhibitor deoxymannojirimycin had no
effect on catalytic activity suggesting that the retention of
glucosylated N-glycans results in the inactivation of this enzyme. The retention of glucosylated N-glycans does not
therefore result in misfolding and degradation of the glycoprotein, as
the enzyme is transported to the melanosome, but may cause
conformational changes in its catalytic domains.
N-Glycans can influence the properties of the protein
to which they are conjugated in a number of different ways (1-3). In many cases N-glycans are required for protein folding,
although there are examples where the addition of N-glycans
to the protein is not required in order for the protein to fold (4).
Each glycoprotein must therefore be studied on a case by case basis to
determine what influence the N-glycan has on the biological and physical properties of a given glycoprotein. One strategy for
studying the role(s) of N-glycosylation involves the use of specific inhibitors of N-linked oligosaccharide processing,
such as -glucosidase I inhibitors (5). In the presence of these compounds the nascent polypeptide is glycosylated co-translationally through the transfer of
Glc3Man9GlcNAc2 from dolichol to
the Asn residue of the Asn-X-Ser/Thr glycosylation sequon.
However, the subsequent enzymatic processing steps, initiated in the
ER1 by the action of
-glucosidase I,
which removes the outer
-1,2-linked glucose residue from the
oligosaccharide, followed by removal of the remaining two glucose
residues in the ER by
-glucosidase II, are prevented (6).
N-Glycosylated glycoproteins are therefore produced that
lack hybrid and complex type oligosaccharide structures. The actions of
inhibitors of
-glucosidases I and II, such as the imino sugar
deoxynojirimycin (DNJ) and its N-alkylated derivatives, have
been extensively characterized in cellular and viral systems (7). In
the presence of these compounds, most N-glycans are arrested
as glucosylated structures and undergo no further processing. However
some glucosylated N-glycans are processed via the Golgi endomannosidase, even in cells treated with high concentrations of
-glucosidase inhibitors (8).
The correct folding of some glycoproteins is regulated within the ER
through the interaction of the unfolded glycoprotein with calnexin and
calreticulin, which have been shown to have chaperone-like functions
(9, 10). Monoglucosylated, partially folded glycoproteins are bound by
calnexin and retained in the ER until correct or complete folding takes
place (11). Glycoproteins bound to calnexin are released through the
action of -glucosidase II and if they are still incompletely folded
can be reglucosylated through the action of an ER-soluble
glucosyltransferase, which only recognizes denatured domains on
glycoproteins (4, 10). The re-glucosylated glycoprotein can then rebind
to calnexin, and folding can continue. Calnexin binds poorly to
glycoproteins carrying three untrimmed glucose residues, as it
preferentially recognizes monoglucosylated species (11). Paradoxically,
-glucosidase inhibitors have minimal effects on cell viability and
secretion (12), and cell lines deficient in
-glucosidases have a
relatively normal phenotype (13). It therefore seems likely that
alternative mechanisms exist within cells to enable the correct folding
of many glycoproteins to occur, when they are prevented from
interacting with their chaperones.
The effects of -glucosidase inhibition on cellular glycoproteins are
selective (14). For example, the transferrin receptor requires correct
oligosaccharide processing for cell surface expression, whereas other
cell surface glycoproteins in the same cell line are expressed
normally (12). The common observation in mammalian systems is that some
glucosylated glycoproteins fold normally (presumably those that are
calnexin-independent or utilize other chaperones during their folding),
whereas others fail to fold completely and are retained in the ER prior
to degradation (15). As yet we have no insight into predicting the fate
of a given glycoprotein when synthesized in a cell treated with
inhibitors of
-glucosidase I. We also have little information
concerning whether a glycoprotein can remain partially misfolded in one
domain of the protein, as a result of retaining glucosylated
N-glycans, while other domains adopt a relatively normal
conformation and are recognized by the appropriate effector molecules
within the cell.
The vast majority of studies to date have focused almost exclusively on
the role of N-glycans on glycoproteins destined for secretion or cell surface expression. However, little information is
available on the behavior of intracellular glycoproteins or glycosylated enzymes in -glucosidase inhibited cells. In this study,
we have investigated the function of an intracellular enzyme when it
carries only glucosylated N-glycans by treating the cells with an inhibitor of
-glucosidase I. We have studied tyrosinase (monophenol, 3,4-
-dihydroxyphenylalanine oxygen oxidoreductase, EC
1.14.18.1), which plays a pivotal role in melanogenesis. It is a single
chain glycoprotein catalyzing the hydroxylation of tyrosine to
-3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPA
quinone (16). Tyrosinase is specifically synthesized within
melanocytes, where it transits through the ER and Golgi and is
transported to specialized intracellular organelles termed melanosomes
(17). The enzymatic activity of tyrosinase is dependent upon the
possession of two copper binding sites. At each of these sites three
histidine residues co-ordinate the copper atom, and both of the copper
atoms co-ordinate an O2 molecule (18). From a total of six
potential glycosylation sites, three are located within or in close
proximity to the active site domains (19).
In this report we demonstrate an almost complete loss of tyrosinase
activity following treatment of B16 mouse melanoma cells with the
-glucosidase I inhibitor N-butyldeoxynojirimycin
(NB-DNJ). Despite the modification in the N-linked
oligosaccharide structure, the enzyme was transported correctly to the
melanosome, but was functionally inactive and therefore failed to
initiate melanin biosynthesis. These results suggest that glucosylated
immature N-glycans do not prevent the transport of
tyrosinase to its specific cellular organelle but that its conformation
may be altered such that there is a profound influence on its enzymatic
activity.
B16-F1 mouse melanoma cells (European Collection of Animal Cell Cultures, Porton Down, United Kingdom (UK)) were cultured in RPMI 1640 medium (Life Technologies, Inc., Paisley, Scotland) containing 10% fetal calf serum (FCS, Sigma, Poole, Dorset, UK), 50 units/ml penicillin, and 50 mg/ml streptomycin (Life Technologies, Inc.), and maintained at 37 °C with 5% CO2. Cells in logarithmic phase growth were washed with phosphate-buffered saline, 0.1 M, pH 7.2 (PBS), released from the plastic with EDTA (5 mM) at 37 °C, washed in PBS, passed through a nylon cell sieve to exclude cell aggregates and resuspended to a density of 1 × 104 cells/ml in culture medium containing NB-DNJ. The NB-DNJ concentration was maintained for the duration of each experiment.
All analytical procedures using B16 cells were standardized to equal cell number using a single cell suspension prepared as described above.
Imino Sugars, Antibodies, and EnzymesNB-DNJ was a gift
from Searle/Monsanto (St. Louis, MO). The rabbit anti-tyrosinase
antiserum (PEP7) (20, 21) was a generous gift from Dr. V. J. Hearing
(NCI, National Institutes of Health, Bethesda, MD).
-Glucosidase I
from rat liver and Aspergillus phoenicis
-1,2-mannosidase
were a gift from Dr. T. Butters (Glycobiology Institute, Oxford, UK).
-Glucosidase II was purified from rat liver microsomes using anion
exchange and gel filtration chromatography, as described previously
(22).
The tyrosine hydroxylase assay has been described previously and measures the 3H2O released as [3,5-3H]tyrosine is hydroxylated to DOPA (16). Briefly, the following reaction mixture (100 µl final volume) was incubated for 60 min at 37 °C: 0.1 M sodium phosphate buffer, pH 7.2, containing 50 µM [3,5-3H]tyrosine (200 mCi/mol, DuPont, Stevenage, Hertfordshire, UK), 5 µM DOPA (as co-factor), and varying concentrations of cell lysate or purified tyrosinase. The reaction mixture was mixed with 950 µl of 0.1 N HCl, 100 mg of Celite (BDH, Poole, Dorset, UK) and 100 mg of charcoal, incubated at 25 °C for 60 min, and centrifuged at 1000 × g for 10 min. The supernatant was retained and 200 µl was scintillation counted. The data were expressed as picomoles of substrate utilized per hour/mg of protein.
DOPA Oxidase AssayThe DOPA oxidase assay measures the second major catalytic reaction of tyrosinase, i.e. the conversion of DOPA to DOPAchrome via DOPA quinone and was performed according to Ref. 23. Briefly, the reaction mixture contained 1 mM DOPA and cell lysate or purified tyrosinase as a source of enzyme in 0.1 M sodium phosphate buffer, pH 7.2, in a total volume of 500 µl and incubated for 30 min at 37 °C. The sample was then measured spectrophotometrically at 475 nm. One unit of tyrosinase was defined as the amount of enzyme catalyzing the oxidation of 1 mmol of L-DOPA to DOPAchrome in 1 min at 37 °C, using the molecular extinction coefficient of DOPAchrome at 475 nm as 3600 (23).
Melanin AssayThe melanin assay measures the production of acid-insoluble melanin from L-[U-14C]tyrosine (100 mCi/mmol, DuPont) and quantifies the entire reaction sequence, including tyrosinase and any post-tyrosinase catalytic activities and was performed according to Ref. 16. Briefly, the reaction was carried out in a total volume of 50 µl of 0.1 M sodium phosphate buffer, pH 7.2, containing 50 µM L-[U-14C]tyrosine, 5 µM DOPA (as co-factor) and varying concentrations of cell lysate or purified tyrosinase as a source of enzyme. After incubation for 60 min at 37 °C the reaction mixture was transferred to Whatman No. 3MM filter disks, washed with 0.1 N HCl and then with acetone, dried, and scintillation counted. Melanin synthesis was expressed as picomoles of tyrosine converted to melanin/h/mg of protein.
Isolation of Melanosome-enriched FractionMelanosomes were isolated from B16 and B16+NB-DNJ cells as described (24). Briefly, the cells were harvested, washed three times with PBS, and lysed with 10 mM phosphate buffer, pH 7.2, containing 0.25 M sucrose. A post-nuclear supernatant was obtained by centrifugation at 700 × g for 15 min at 4 °C and further fractionated by differential centrifugation at 11,500 × g for 30 min at 4° C, yielding a melanosome-enriched pellet.
Metabolic Labeling with [35S]Methionine/CysteineB16 cells were harvested with EDTA, washed three times with PBS, and resuspended in methionine/cysteine-free RPMI 1640 medium (Life Technologies, Inc.) supplemented with 1% dialyzed FCS. Cells (107 cells/ml) were preincubated in the presence or absence of NB-DNJ for 1 h before the addition of 200 µCi/ml [35S]methionine/cysteine (Tran35S-label, >1100 Ci/mmol, ICN Flow, Thame, Oxfordshire, UK) for 4 h. Following harvesting and washing with PBS, cells were either lysed in 1% Nonidet P-40 in 10 mM phosphate buffer, pH 7.2, containing 100 µM phenylmethylsulfonyl fluoride, or suspended in 0.25 M sucrose to isolate melanosomes as described. The melanosome fractions were lysed in the same buffer as the cells.
Metabolic Labeling with [2-3H]MannoseB16 cells (106 cells/ml) were suspended in RPMI 1640 medium (without glucose) supplemented with 1 mM sodium pyruvate and 1% dialyzed FCS and preincubated in the presence or absence of NB-DNJ. After 1 h, 500 µCi/ml [2-3H]mannose was added, and cultures were labeled for 24 h. Following harvesting and washing with PBS, cells were lysed in 1% Nonidet P-40 (Sigma) in 10 mM phosphate buffer, pH 7.2, containing 100 µM phenylmethylsulfonyl fluoride.
Immunoprecipitation35S-Labeled or unlabeled
cell or melanosomal lysates were incubated with PEP7 antiserum at 10 µl/100 µl of lysate for 1 h at 37 °C followed by the
addition of 50 µl of protein G-Sepharose (Pharmacia Biotech Inc.,
Upsalla, Sweden) and further incubation for 30 min at room temperature.
The slurry was washed five times with 0.1% Nonidet P-40 in 10 mM phosphate buffer and once in water. Tyrosinase was
eluted by boiling for 5 min in SDS sample buffer with 5%
2-mercaptoethanol. For chromatographic analysis,
[2-3H]mannose-labeled tyrosinase was isolated from cell
lysates as described above and further purified by preparative
electrophoresis in 7.5% SDS-PAGE. [2-3H]Mannose-labeled
oligosaccharides were released from tyrosinase by PNGase F (New England
Biolabs, Hitchin, Hertfordshire, UK) digestion (0.1 unit/1 mg of
protein) in SDS-PAGE reducing buffer, containing 1% 2-mercaptoethanol
and 0.9% SDS, for 48 h at 37 °C. Released oligosaccharides
were desalted by passing the reaction mixture over a column of 100 µl
each of Chelex 100, Dowex AG50 X-12 (H+ form), Dowex AG3
X-4A (OH
form). The eluate was pooled with a wash of 5 bed volumes of water, filtered, and concentrated before separation by
gel permeation.
Enzymatically released radiolabeled oligosaccharides were subjected to high resolution gel permeation chromatography using two Bio-Gel P-4 (1.5 × 100 cm) columns in series. The columns were maintained at 55 °C and eluted with water, and fractions (0.5 ml) were monitored for radioactivity using liquid scintillation counting. Elution positions in glucose units were determined by simultaneous separation of a ladder of partially hydrolyzed dextran and detected on the basis of refractive index.
Electrophoresis and Western BlottingSDS-PAGE was performed
as described by Laemmli (25) in 7.5% acrylamide gels. The samples were
mixed 2:1 (v/v) with sample buffer (0.18 M Tris/HCl, pH
6.8, containing 15% glycerol, 9% SDS). To detect specific tyrosinase
activity after electrophoresis, gels were incubated for 30 min at
37 °C with 0.1 M phosphate buffer, pH 6.8, containing 2 mM L-DOPA and 4 mM 3-methyl-2-
benzothiazolinone hydrazone, as described previously (26). For Western
blotting, cell or melanosomal lysates were separated by electrophoresis under denaturing conditions (sample was mixed 2:1 (v/v) with sample buffer containing 0.18 M Tris/HCl, pH 6.8, 15% glycerol,
9% SDS, 10% 2-mercaptoethanol and boiled for 5 min), transferred to
Immobilon membrane (Amersham International, Amersham, UK) and reacted
with PEP7 antiserum (diluted 1:250). Visualization of antibodies was performed using the ECL chemiluminescent detection system (Amersham). Deglycosylated samples for Western blotting were prepared by digestion with endo H or PNGase F. Digestion of crude extracts with endo H was
performed following denaturation of samples in 0.15 M
citrate buffer, pH 5.5, containing 2% SDS and 5% 2-mercaptoethanol
for 5 min at 100 °C. Samples were cooled, diluted twice with water, 10 units/ml endo H (New England Biolabs) was added, and digestion performed for 24 h at 37 °C. For PNGase F digestion, samples
were denatured by heating (5 min at 100 °C) in the presence of SDS reducing buffer (10 µl/100 µl of sample) and 0.1 unit/ml PNGase F
added and incubated for 24 h at 37 °C.
B16 cells were harvested, washed in
PBS, and resuspended at a density of 1 × 105/ml and
100 µl of the cell suspension spun onto a Cytospin slide (Cytospin,
Shandon Scientific, Cheshire, UK). The slides were air-dried and
acetone-fixed (30 s, ice-cold acetone), blocked in 5% FCS in PBS (30 min, room temperature in a humidified chamber), the blocking agent was
removed, and the slides were incubated with the rabbit PEP7
antiserum (1:100 in PBS/5% FCS, for 30 min at room temperature). The
slides were washed three times in PBS and incubated as described above
with anti-rabbit fluorescein isothiocyanate (Sigma). The slides were
washed as described and mounted in Vectashield (Vector Laboratories,
Burlington, CA) and examined by fluorescence microscopy (Zeiss,
Axioplan).
B16 cells were cultured on sterile glass coverslips in individual wells of a 24-well plate (Costar) for 3 days in the presence or absence of 0.5 mM NB-DNJ. The cells were harvested, washed in PBS, fixed in ice-cold acetone for 30 s, air-dried, and immunostained as described for fluorescence microscopy (see above), except the secondary antibody used was anti-rabbit Texas Red. The cells were examined using a Bio-Rad MRC 1024 confocal microscope equipped with a 15-milliwatt crypton argon laser. The images were prepared using Lasersharp 2.1 and Adobe Photoshop 3.0. The scale bar represents 10 µM.
Electron MicroscopyMelanosome-enriched fractions were generated as described from B16 and B16+NB-DNJ, fixed in 4% glutaraldehyde (Sigma) in 0.1 M phosphate buffer, pH 7.2, for 1 h at room temperature. The fractions were pelleted, washed three times with phosphate buffer, embedded in 4% agar, cut into 1-mm cubes, and fixed in 1% osmium tetroxide (Sigma). The samples were dehydrated through an ethanol series followed by propylene oxide and embedded in Epon 812 (Sigma). The sections were stained with uranyl actetate/lead citrate and examined with a Tesla-500 electron microscope.
B16 cells were
cultured in the presence (B16+NB-DNJ) or absence (B16) of
50 µM or 500 µM NB-DNJ for 3 days. When the
cells were visually inspected the untreated cells exhibited normal
melanin pigmentation, whereas 500 µM NB-DNJ treatment
resulted in nonpigmented cells. B16 cells treated with 50 µM NB-DNJ exhibited an intermediate degree of melanin
content (Fig. 1A). When the two tyrosinase
isoenzyme activities were visualized on gels following electrophoresis
of crude extracts derived from NB-DNJ treated or untreated cells, tyrosinase activity could only be detected in extracts from untreated cells (Fig. 1B). Both activities of tyrosinase (tyrosine
hydroxylase and DOPA oxidase) were profoundly inhibited by NB-DNJ
treatment, and this inhibition was dose-dependent (Fig.
2A). When cells were treated with 500 µM NB-DNJ tyrosine hydroxylase, activity decreased by
85%, DOPA oxidase activity by 95%, and melanin content decreases by
98% relative to untreated cells (Fig. 2A). The inhibition
of tyrosinase activity and melanin biosynthesis was observed within 24 h following NB-DNJ treatment (Fig. 2B). Total
reversibility of tyrosinase activity and melanin biosynthesis were
observed when NB-DNJ was washed out of the cultures (Fig.
2C). The effect of NB-DNJ on tyrosinase activity was found
to be indirect. This was demonstrated by incubating untreated cells
lysates with NB-DNJ (0.5 mM) for 2 h prior to the DOPA
oxidase assay. No significant inhibition of tyrosinase activity was
observed (Table I). The inhibition of melanin
biosynthesis was found to correlate with treatment of B16 cells with
known inhibitors of -glucosidases I and II such as castanospermine,
DNJ, and N-methyl-DNJ (data not shown), whereas the
mannosidase inhibitor DMJ failed to inhibit tyrosinase activity and did
not inhibit melanin biosynthesis (Table I). The specific activity of
tyrosinase was found to be very similar in the total cell lysate from
B16 cells and the melanosomal fractions (Table I).
Kinetics of tyrosinase activities and melanin synthesis inhibition during incubation of B16 cells with NB-DNJ. A, cells were incubated for 72 h in the presence or absence of different concentrations of NB-DNJ and cell lysates analyzed for enzymatic activity and melanin content. B, cells were incubated for 72 h in the presence or absence of 0.5 mM NB-DNJ and cell lysates analyzed for enzymatic activity and melanin content. C, cells were incubated for 72 h in the presence or absence of 0.5 mM NB-DNJ. NB-DNJ was washed out of the cultures, and the cells were cultured for the times indicated. All assays were performed in triplicate, and the standard deviations fell within ±10% of the mean.
|
Cells were
metabolically labeled with [35S]methionine/cysteine for
4 h and immunoprecipitated with anti-PEP7 antiserum. A 72-kDa species was precipitated from B16 cells and a 69-kDa species from B16+NB-DNJ cells (Fig. 3A). Based
on cell lysates obtained from the same number of B16 and
B16+NB-DNJ cells, the level of B16+NB-DNJ
tyrosinase detected by immunoprecipitation was unchanged by NB-DNJ treatment. To determine whether or not the tyrosinase detected in
whole cell lysates from B16+NB-DNJ cells was
localized to the melanosome, a melanosome-enriched fraction was
generated by differential centrifugation (24).
The pellet containing the melanosomes from untreated cells was darkly
pigmented (black) while the supernatant remained clear indicating that
the pellet contained melanosomes. The same fraction from NB-DNJ-treated
cells was lightly pigmented reflecting the loss of melanin resulting
from drug treatment. This was further confirmed by electron microscopy
on the melanosome-enriched fraction (Fig. 3B). As
anticipated this fraction contained melanosomes at various stages of
development, as indicated by their degree of pigmentation and their
heterogeneous size. The fraction from B16+NB-DNJ contained
organelles of comparable size to those from untreated cells, but they
were less electron-dense, indicating reduced levels of melanin. Similar
results to those observed with whole cell lysates were obtained by
immunoprecipitating tyrosinase from the melanosomal enriched fraction
of B16 and B16+NB-DNJ cells (Fig. 3A),
indicating the presence of the enzyme in the melanosome. Tyrosinase
expression was further assessed by Western blotting. As presented in
Fig. 4A, identification of tyrosinase from
melanosomal extracts with anti-tyrosinase antibodies showed the same
decrease in B16+NB-DNJ tyrosinase molecular weight compared
with the B16 enzyme in the immunoprecipitation experiments. Treatment
of B16 and B16+NB-DNJ tyrosinase with PNGase F, which
hydrolyzes the -aspartylglycosylamine bond between Asn and the
innermost GlcNAc of the N-glycan, yielded a 60-kDa
polypeptide for both enzymes (Fig. 4A). PNGase F digestion showed that the B16+NB-DNJ tyrosinase was synthesized as a
60-kDa polypeptide chain and co-/post-translationally modified to give
the mature 69-kDa glycoprotein. Moreover, extraction of melanosomal
membrane proteins with Triton X-114 followed by Western blotting
demonstrated that B16+NB-DNJ tyrosinase was localized to
the melanosomal membrane (Fig. 4B). The same localization
was revealed for B16 tyrosinase (Fig. 4B), in good agreement
with previous reports demonstrating that tyrosinase is an integral
membrane glycoprotein (27). Localization of the enzyme was confirmed by
immunocytochemistry (Fig. 5), showing that there was no
qualitative or quantitative difference in the general location of
tyrosinase in B16 and B16+NB-DNJ cells. We examined the
distribution of tyrosinase at higher resolution by confocal microscopy
(Fig. 6) and observed a punctate cytoplamic pattern of
staining in both B16 and B16+NB-DNJ, confirming the
melanosomal localization of tyrosinase, irrespective of NB-DNJ
treatment.
Structural Analysis of B16+NB-DNJ Tyrosinase Oligosaccharides
Incubation of B16 and B16+NB-DNJ
tyrosinases with endo H resulted in changes in the electrophoretic
mobilities of both glycoproteins. While B16+NB-DNJ
tyrosinase was completely sensitive to endo H, B16 tyrosinase was only
partially sensitive, suggesting that tyrosinase normally contains both
high mannose and complex type oligosaccharides (Fig. 4A).
Increased endo H sensitivity was also observed with tyrosinase derived
from DMJ-treated cells (Fig. 4A). The status of the
tyrosinase N-glycans from B16 and B16+NB-DNJ
cells was investigated further by labeling the cells with
[2-3H]mannose for 24 h and B16 and
B16+NB-DNJ tyrosinases isolated from the cell lysates as
described. Release of [2-3H]mannose-labeled unreduced
oligosaccharides from the purified enzymes was achieved by PNGase
digestion. Gel permeation chromatography was used to determine the size
profile of the glycan pools (nonsialylated oligosaccharides). The
profile obtained from B16 tyrosinase contained a mixture of at least
nine different oligosaccharide structures, eluting between 19.9 and 7.5 glucose units (Fig. 7). This is consistent with the
presence of a mixture of complex (19.9-15.8 glucose units) and
oligomannose (12.3-7.5 glucose units) oligosaccharides on B16
tyrosinase. A similar composition was reported for hamster melanoma
tyrosinase with both complex type N-glycans and
oligomannose (Man3-9 GlcNAc2) structures being
present (23). Analysis of B16+NB-DNJ tyrosinase glycans
(Fig. 8A, peaks A-E) identified five major peaks of 15.3, 14.3, 13.3, 12.3, and 11.8 glucose units. There was a
trend toward larger oligosaccharide structures in the
B16+NB-DNJ profile, relative to the profile derived from
B16 tyrosinase N-glycans (Fig. 7). The
B16+NB-DNJ tyrosinase oligosaccharides were digested with a
mixture of -glucosidases I and II to determine their glucosylation
status. Only three major peaks were observed following digestion
(peak F, 12 glucose units; peak G, 11.3 glucose
units; peak H, 10.2 glucose units), corresponding to a
3-glucose unit reduction of peaks A-C, consistent with the loss of three glucose moieties (Fig. 8B). Peaks
F-H were pooled, digested with A. phoenicis
-1,2-mannosidase, and re-chromatographed on Bio-Gel P-4. The profile
showed a single species corresponding to the
Man5GlcNAc2 structure (Fig. 8C). The
sequential glycosidase digestions described are consistent with
structures of oligosaccharides in peaks A, B, and
C, being Glc3Man9,
Glc3Man8, and Glc3Man7, respectively (Fig. 9).
Effect of Deglucosylation on B16+NB-DNJ Tyrosinase Activity
Purified B16+NB-DNJ tyrosinase was
deglucosylated by digestion with a mixture of -glucosidases I and II
and assayed for DOPA oxidase activity (Fig. 10). The
anticipated increase in electrophoretic mobility of
B16+NB-DNJ tyrosinase was observed post-glucosidase
digestion, demonstrating that the glucose residues had been removed.
The tyrosinase post-glucosidase digestion was always observed to be
more immunoreactive with the anti-tyrosinase antibody on Western blots,
suggesting that there was increased exposure of the C-terminal epitope
recognized by this antiserum following de-glucosylation (Fig. 10). As
shown in Table I, the deglucosylated B16+NB-DNJ tyrosinase
showed the same enzymatic activity as glucosylated B16+NB-DNJ tyrosinase.
In this report we have examined the effects of inhibiting
N-glycan processing, using the -glucosidase inhibitor
NB-DNJ, on the biological properties of murine tyrosinase. We have
found that, in contrast to some other glucosylated glycoproteins (12, 15), the enzyme does not misfold in the presence of glucosylated N-glycans to a degree that causes it to be retained in the
ER and degraded. Instead it is transported to its correct cellular location, namely the melanosome. Indeed, the amounts of tyrosinase immunoprecipitated from the melanosomes of B16 and
B16+NB-DNJ cells are comparable, showing that similar
levels of B16 and B16+NB-DNJ tyrosinase are synthesized and
transported to the correct organelle, irrespective of their
N-glycan composition. This implies that either the folding
of the protein in regions required for transport must be comparable
with the native molecule or that the transport is independent of its
folding. However, despite correct localization this enzyme has
virtually no catalytic activity in B16+NB-DNJ cells, and
therefore the cells cannot support melanogenesis, and they are
profoundly deficient in pigment relative to B16 cells.
Tyrosinase is a bifunctional enzyme in that it catalyzes the
hydroxylation of tyrosine to DOPA (the rate-limiting step in melanin
biosynthesis) and the oxidation of DOPA to DOPA quinone. For the first
step, tyrosinase requires DOPA as a co-factor, and the enzyme has been
proposed to contain two catalytic sites involved in the binding of
tyrosine and DOPA, respectively (16). Tyrosine hydroxylase activity in
NB-DNJ-treated B16 cells is diminished to 15% of its normal activity
and DOPA oxidase activity to 5%, indicating that both catalytic sites
have been affected. Upon removal of NB-DNJ from the culture medium the
cells re-synthesize tyrosinase, and therefore both activities (tyrosine
hydroxylase and DOPA oxidase) of the enzyme can be detected in cell
extracts within 24-72 h. Tyrosinase dysfunction in
B16+NB-DNJ cells is also indicated by the marked
de-pigmentation of these cells due to the incapacity of the
B16+NB-DNJ tyrosinase to initiate melanin biosynthesis.
Conversely, others have reported that the overall activity of mouse
tyrosinase is not inhibited when B16 cells are incubated (for 11-25
days) with glucosamine, a weak glucosidase inhibitor, despite the loss
of pigmentation (28, 29). These results, obtained by monitoring enzyme
activity, were interpreted as an indication that some isoforms of
tyrosinase fail to reach the melanosome. In contrast,
immunoprecipitation and Western blotting experiments, performed in this
study, have shown that B16+NB-DNJ tyrosinase is correctly
transported to the melanosomes. Further support comes from the report
of the effects of -glucosidase inhibitors on human tyrosinase (30).
The enzyme was still synthesized in the human cell line MM96E, but had
no activity and therefore the cells were nonpigmented (30). This is
therefore analogous to our findings with NB-DNJ-treated B16 mouse
melanoma cells. Moreover, Triton X-114 extraction of the
B16+NB-DNJ cells demonstrated that B16+NB-DNJ
tyrosinase is correctly incorporated in the melanosomal membrane in an
analogous fashion to the B16 tyrosinase. For melanogenesis this is an
important observation, as it is known that even if the enzyme is active
immediately after completion of its synthesis, melanin synthesis is
only initiated after tyrosinase reaches the melanosomal membrane (27).
Correct insertion of B16+NB-DNJ tyrosinase into the
membrane suggests that the de-pigmentation observed in B16+
cells is due to inactivity of tyrosinase, rather than its incorrect transport. In addition, the same low level of activity of
B16+NB-DNJ tyrosinase was found both in melanosomal and
microsomal fractions, suggesting that inactivation does not occur
during, or as a consequence of, defective transport. On the other hand,
direct incubation of untreated cell lysates with NB-DNJ results in the
detection of normal levels of tyrosinase activity. This shows that
NB-DNJ does not act as a direct inhibitor of this enzyme but that it causes an indirect effect on tyrosinase activity, presumably through the alteration in N-glycan structure. We conclude that
B16+NB-DNJ tyrosinase is therefore synthesized in an
inactive form. Digestion with PNGase F revealed that
B16+NB-DNJ tyrosinase is synthesized as a 60-kDa
polypeptide. The same molecular weight was obtained for PNGase-treated
B16 tyrosinase. This is in agreement with the predicted polypeptide
molecular weight based on the tyrosinase cDNA, which predicts 533 amino acids, corresponding to a molecular mass of 58-60 kDa (19).
Following co- and post-translational attachment of carbohydrates, the
molecular mass of B16 tyrosinase increases to approximately 72 kDa
(19). Mature B16+NB-DNJ tyrosinase has a molecular mass of
only 69 kDa, 3 kDa less than B16 tyrosinase. This indicates that the
differences in molecular mass appear to be at the post-translational
level and are related to differences in the oligosaccharide moiety.
Analysis of B16+NB-DNJ tyrosinase oligosaccharide sequences revealed the existence of glucosylated oligosaccharides, including Glc3Man9, Glc3Man8,and Glc3Man7, which is in agreement with the N-glycan structures found on gp120 expressed in the presence of NB-DNJ (31). The small percentage of more processed N-glycan structures observed could explain the low level of residual tyrosinase activity observed in this study and may result from the action of the Golgi endomannosidase (8). Our results show that the untreated tyrosinase has a mixture of high mannose and complex type oligosaccharides. Similar N-glycan profiles have been reported for hamster tyrosinase (23),suggesting that there is a degree of conservation of tyrosinase N-glycan structures among at least two mammalian species.
To determine whether the lack of complex N-glycans was
sufficient to render tyrosinase inactive, B16 cells were treated with DMJ, an inhibitor of Golgi -1,2-mannosidase. Tyrosinase was rendered partially sensitive to endo H following treatment of B16 cells with
DMJ. However, there was no effect on tyrosinase activity, suggesting
that the absence of complex type N-glycan structures is not
responsible for the maintenance of tyrosinase in an active form.
Furthermore, these data suggest that retention of glucosylated high
mannose structures, due to NB-DNJ treatment, directly results in the
loss of tyrosinase activity. This is not due to the glucosylated N-glycan directly interfering with the catalytic site as was
shown by removing the three glucose residues, which did not restore enzyme activity. The loss of activity therefore may result from partial
misfolding of the catalytic site, possibly showing that folding of this
domain is chaperone dependent. It is interesting to note that the
transport to the melanosome remains normal. Perhaps this illustrates
that the overall conformation of this protein occurs by independent
domain folding mechanisms.
Similar conclusions have been drawn from studies of human
immunodeficiency virus glycoproteins expressed in NB-DNJ-treated Chinese hamster ovary cells (32). The structure of gp120 was probed
using a panel of over 40 monoclonal antibodies. It was found that most
of the regions of gp120 expressed in the presence of NB-DNJ
(gp120+) were indistinguishable from gp120 expressed in the
absence of the compound (gp120). Furthermore, the
gp120+ retained its ability to bind to CD4 with an affinity
comparable with gp120
. However, when the conformation of
the V1/V2 loops were investigated, it was found that this region was
affected by the retention of glucosylated N-glycans. This
alteration in antibody recognition was attributed to a change in the
conformation of this region of the molecule. It is of interest that
gp120+ transits through the ER and Golgi and is secreted
from the cell at comparable rates relative to gp120
, yet
has localized conformational changes due to retention of glucosylated
N-glycans during protein folding (32). We conclude that the
retention of glucosylated N-glycans results in the loss of
catalytic activity but does not impair intracellular transport. As a
consequence, melanogenesis is blocked due to the pivotal nature of this
enzyme in the melanin biosynthetic pathway. Inactivation of tyrosinase
may result from the failure of the enzyme to bind copper either through
changes in the conformation of the active site in the presence of
glucosylated N-glycans or indirectly due to impaired copper
transport within the cell. Another possibility is that the protein
undergoes independent domain folding with the active site conformation
being critically dependent on the interaction of the protein with
chaperones during its folding. The mechanism(s) of tyrosinase
inactivation in NB-DNJ-treated cells is currently under
investigation.
We thank Searle/Monsanto for NB-DNJ, Vincent
Hearing for the generous gift of the PEP7 antiserum, Terry Butters
for
-glucosidases I and II and for helpful discussions, Mark Wormald
for discussions on protein folding, Gabrielle Neises for help with the
EM, and Bryan Mathews for his expert technical assistance with
carbohydrate analysis.