From the Christian Albrechts Universität zu Kiel, Biochemisches Institut, Olshausenstrasse 40, D-24098 Kiel, Germany
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ABSTRACT |
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The critical step in lysosomal targeting of
soluble lysosomal enzymes is the recognition by an
UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The
structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our
current knowledge is largely based on the introduction of targeted
amino acid substitutions into lysosomal enzymes and analysis of their
effects on phosphotransferase recognition. We have investigated the
effect of eight anti-arylsulfatase A monoclonal antibodies on the
interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a
lysine-rich surface area of arylsulfatases A and B is essential for
proper recognition by the phosphotransferase. Monoclonal antibodies
bind to at least six different epitopes at different locations on the
surface of arylsulfatase A. All antibodies bind outside the lysine-rich
recognition area, but nevertheless Fab fragments of these antibodies
prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but
involves the simultaneous recognition of large parts of arylsulfatase A.
The correct lysosomal targeting of soluble lysosomal enzymes
mainly depends on the synthesis of mannose 6-phosphate residues on
their N-linked oligosaccharide side chains. These mannose
6-phosphate residues are lysosomal targeting signals. In the late Golgi
compartments, lysosomal enzymes bind to mannose 6-phosphate receptors,
which mediate the vesicular transport to the lysosomes (for review, see
Refs. 1 and 2). The synthesis of mannose 6-phosphate residues is a
two-step process: in the first reaction the
UDP-N-acetylglucosamine:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase (EC 2.7.8.17) (briefly called
phosphotransferase1)
transfers N-acetylglucosamine-1-phosphate from
UDP-N-acetylglucosamine to mannose residues of high
mannose-type oligosaccharide side chains of lysosomal enzymes yielding
N-acetylglucosamine-1-phospho-6-mannose residues. In a
second reaction the N-acetylglucosamine is removed by a
phosphodiesterase generating mannose 6-phosphate residues on the
oligosaccharide side chains (1). The phosphotransferase has been
purified to homogeneity (3). It is a large enzyme of two disulfide
linked homodimers of 166 and 51 kDa and two noncovalently linked 56-kDa
subunits. The holoenzyme has a molecular mass of 540 kDa (4).
The specific recognition of soluble lysosomal enzymes by the
phosphotransferase is the critical step for proper intracellular targeting of these enzymes. Thus, the phosphotransferase must be able
to specifically recognize lysosomal enzymes, but since these represent
a heterogeneous family of proteins, it must also be able to interact
with structurally very diverse enzymes. No obvious sequence
similarities suggesting a common homologous targeting signal can be
detected among lysosomal enzymes. Earlier studies demonstrated that the
specific recognition of lysosomal enzymes by the phosphotransferase
depends on the native conformation of lysosomal enzymes (5). In recent
years, various studies tried to characterize the recognition
determinants of lysosomal enzymes employing in vitro
mutagenesis strategies. In the COOH-terminal lobe of lysosomal protease
cathepsin D, two regions have been delineated to be a minimal
determinant for recognition by the lysosomal enzyme phosphotransferase,
a lysine in position 203 and a stretch of 32 amino acid close to the
COOH terminus (6). Crystallographic structure determinations revealed
that those critical residues are in close proximity on the surface of
cathepsin D (7). However, further studies showed that regions in the NH2-terminal lobe of cathepsin D also contribute to
efficient recognition by the phosphotransferase (8-10). These studies
demonstrated that various distantly spaced residues may act
cooperatively to yield the fully effective sorting signal. Similar
conclusions have been reached for aspartylglucosaminidase, in which
three lysines distantly spaced on the enzyme surface contribute to the proper recognition by the phosphotransferase (11), and for cathepsin D
and L in each of which two lysines approximately 34 Å apart are
critical recognition determinants (12).
A number of studies on cathepsin D (6, 8-10, 12), cathepsin L (12,
13), DNase I (14), and aspartylglucosaminidase (11) indicated that
recognition of lysosomal enzymes depends on lysines as critical
residues. Besides the identification of lysines as a common feature of
the lysosomal enzyme recognition determinant, its overall structure
remains unknown. Comparisons of three-dimensional structures of
recently crystallized lysosomal enzymes suggested that a loose
consensus sequence in a Arylsulfatases A (EC 3.1.6.8) and arylsulfatase B (EC 3.1.6.12) are
lysosomal enzymes involved in the degradation of sulfated glycolipids
and glycosaminoglycans, respectively. Arylsulfatase A is synthesized as
a 62-kDa polypeptide, which bears three high mannose-type
oligosaccharide side chains, two of which are phosphorylated by the
phosphotransferase (16, 17). The position of the recognition determinant in lysosomal arylsulfatase A and homologous sulfatases is
unknown. Since our current knowledge on the requirements of the
interaction of the lysosomal enzymes with the phosphotransferase is
based on the introduction of targeted amino acid substitutions we have
chosen to characterize phosphotransferase recognition determinant on
arylsulfatase A with monoclonal antibodies. Recently, we reported the
generation of four monoclonal antibodies specific for human
arylsulfatase A (18). Fab fragments of these antibodies inhibited the
synthesis of mannose 6-phosphate residues on arylsulfatase A in an
in vitro phosphorylation system. Epitope mapping revealed that these antibodies recognized identical or adjacent epitopes on the enzyme, and it was assumed that the epitope was part of or close
to the phosphotransferase recognition determinant. In order to extend
this study and to map the recognition determinant more precisely,
we have generated eight additional antibodies and examined their
ability to interfere with mannose phosphorylation.
Materials--
Cell culture media RPMI 1640 for hybridomas and
Dulbecco's modified Eagle's medium for all other cell types were from
Life Technologies, Inc. Fetal calf serum was obtained from Biochrom. DNA digesting and DNA modifying enzymes were from New England Biolabs
or Life Technologies, Inc. [ Production of Monoclonal Antibodies--
Female BALB/c mice 6 weeks of age were injected three times (day 1, 14, and 28)
intraperitoneally with 100 µg of arylsulfatase A. Recombinant enzyme
was purified by immunoaffinity purification as described earlier (18).
Prior to injection the enzyme was adsorbed to aluminum hydroxide.
Anti-arylsulfatase A titers were monitored 12 days after the last
immunization. Animals scoring positive were boosted with 50 µg of
arylsulfatase A and sacrificed 3 days later. Spleen cells of mice were
fused with X63-Ag8.653 myeloma cells. Fusion was done as described
(21). After fusion, cells were plated in 200 wells of 24-well hybridoma
plates (Greiner) in RPMI 1640 supplemented with 10% fetal calf serum,
penicillin, streptomycine, 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine
(Boehringer-Mannheim). The medium was further supplemented with 10%
conditioned medium from the J774 cell line. Wells containing anti-arylsulfatase A immunoglobuline secreting hybridomas were identified via a radioimmunoassay in which an iodinated secondary anti-mouse light chain antibody raised in rabbits was used (Dakopatts, Copenhagen). Clones in positive cells were subcloned and reanalyzed by
radioimmunoassay. Immunoglobulin subclass determination was performed
with a kit from Boehringer-Mannheim. MAbs A3, C, E, and F are
IgG1; mAbs A1, A2, and A4 are IgG2a; and A5 is
IgG2b. All antibodies have
In part of the experiments three previously published mAbs have been
used (18). To facilitate reading of this article these antibodies have
been renamed, 19C2 is B1, 20D2 is B2, and 11B5 is D.
Purification of Monoclonal Antibodies and Fab
Fragments--
Hybridomas were cultured in the Hereaus Miniperm
system, which in all cases yielded several milligrams of mAbs.
IgG2 mAbs were purified via affinity chromatography on
Protein A-Sepharose and IgG1 on Protein G-Sepharose
(Pharmacia). Affinity chromatography was performed according to the
manufacturers protocols. For production of Fab fragments, purified
antibodies were adjusted to a concentration of 1-5 mg in
phosphate-buffered saline, 5 mM cysteine. Papain was added
at a molar ratio of 1:40 mol of IgG. Depending on the antibody,
proteolysis was performed at 37 °C for 1 to 4 h. Reaction was
terminated by the addition of iodoacetamide to a final concentration of
20 mM. Fab fragments were purified by gel filtration on a
Superdex G-75 column (Pharmacia) equilibrated with phosphate-buffered saline.
Immunoprecipitation, SDS-PAGE, and Western
Blots--
Immunoprecipitation of unlabeled arylsulfatase A under
nondenaturing conditions was performed in 50 mM Tris/HCl,
pH 7.4, 0.15 M NaCl, 0.05% Triton X-100. Five to 10 milliunits of arylsulfatase A were incubated with 10 µg of antibodies
for 12-16 h. To enhance precipitation, 10 µg of goat anti-mouse
affinity purified antibodies (Dianova) were added for another 2 h
before Ig were collected by the addition of 60 µl of
Staphylococcus aureus suspension (Pansorbin, Calbiochem).
After 30 min of continuous shaking, bacteria were pelleted for 4 min at
10,000 × g. Pellets were resuspended in immunoprecipitation buffer and washed once. Arylsulfatase A activity was determined in supernatants and pellets.
Monoclonal Antibody Competition Assay--
96-well enzyme-linked
immunosorbent assay plates were coated with 125 ng of mAbs and
subsequently blocked with an excess of casein. Simultaneously,
arylsulfatase A was incubated with a 10-fold molar excess of mAb in 20 mM Tris/HCl, pH 7.4, 0.15 M NaCl.
MAb-arylsulfatase A complexes generated in liquid phase were then added
to the mAb-coated wells and allowed to bind for 2 h at 37 °C.
Afterward liquid phase was removed, the wells were washed twice, and
arylsulfatase A bound to the wells was quantified by activity
determination (18). If the antibody coupled to the well and complexed
to the arylsulfatase A in the liquid phase compete and thus cannot bind
arylsulfatase A simultaneously, the enzyme cannot be retained in the wells.
Detection of Octamerization of Arylsulfatase A--
96-well
enzyme-linked immunosorbent assay plates were coated with 100 ng of
arylsulfatase A/well for 16 h at 4 °C. Purified arylsulfatase A
can be labeled via incubation with
[35S]cysteine.2
35S-Labeled arylsulfatase A was incubated with a
10-fold molar excess of mAbs. The antibody complexed with
[35S]arylsulfatase A was added to the wells at neutral pH
when octamerization of arylsulfatase A does not occur. Then pH of the
wells was titrated to pH 4.5 and incubation continued for 1 h. Low
pH favors octamerization of arylsulfatase A so that arylsulfatase
A-antibody complexes should bind to solid phase arylsulfatase A if the
antibody does not interfere with octamerization. Liquid phase was
removed and wells were washed with 50 mM sodium acetate
buffer, pH 4.5. Radioactivity bound to the plates was determined. As a
control a duplicate plate was washed with neutral buffer, which
reverses association of [35S]arylsulfatase A with
arylsulfatase A bound to the wells.
In Vitro Phosphorylation of Arylsulfatase A--
Preparation of
Golgi membranes, [ Chemical Modification of Amino and Carboxyl Residues--
To
modify carboxyl residues 50 µg of arylsulfatase A or arylsulfatase B
were dialyzed against 20 mM sodium acetate, pH 5.2, and
samples were divided into two parts of 50 µl each. One part received
no addition and served as a control, the other part was supplemented
with 20 mM
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydroxychloride
(EDC) and 100 mM ethylendiamide. After incubation for
20 min at 30 °C, the reaction was stopped by increasing sodium acetate concentration to 0.66 M. Treated and untreated
samples were subsequently dialyzed against Tris-buffered saline.
To modify amino groups of lysines, 50 µg of arylsulfatase A or
arylsulfatase B were dialyzed against phosphate-buffered saline, pH
8.0, and samples were split into two aliquots. One aliquot received no
additions and to the other 17.5 mM
sulfo-N-hydroxy-succinimidyl acetate (SNA) was added.
Incubation was for 90 min on ice and reaction was terminated by
dialysis against Tris-buffered saline. In each case, the success of the
modification reaction was monitored by alterations in isoelectric
focussing pattern.
Characterization of Anti-arylsulfatase A Monoclonal
Antibodies--
Mice were immunized with recombinant human
arylsulfatase A and a total of 8 stable hybridomas secreting
anti-arylsulfatase A antibodies were obtained. These antibodies
precipitated arylsulfatase A under nondenaturing conditions from
extracts of Ltk Anti-arylsulfatase A Monoclonal Antibodies Inhibit in Vitro
Phosphorylation of Arylsulfatase A--
The synthesis of mannose
6-phosphate residues, which represent the specific lysosomal targeting
signal can be performed in vitro (18, 22). Purified
lysosomal enzymes are incubated with a Golgi fraction as a source
of lysosomal enzyme phosphotransferase and
[
Fig. 1 shows an experiment in which
recombinant arylsulfatase A purified to apparent homogeneity was
phosphorylated in vitro in the absence or presence of
control or anti-arylsulfatase A mAbs. Addition of an irrelevant control
mAb has no effect on arylsulfatase A phosphorylation, whereas all mAbs
(top panel) or their Fab fragments (bottom panel)
inhibited phosphorylation almost completely. Addition of
anti-arylsulfatase A mAbs or Fab fragments did not inhibit phosphorylation of arylsulfatase B, which was co-phosphorylated in the
same sample, excluding nonspecific inhibition of phosphorylation by the
addition of anti-arylsulfatase A mAbs.
Inhibition of arylsulfatase A phosphorylation may be explained in two
ways. 1) The antibodies inhibit recognition of arylsulfatase A by the
phosphotransferase, because they mask the determinant responsible for
lysosomal enzyme recognition. In this case the arylsulfatase A-mAb
complexes will not be able to interact with the phosphotransferase. 2)
The antibodies may still allow arylsulfatase A to be recognized by the
phosphotransferase as a lysosomal enzyme, but after binding,
phosphorylation of oligosaccharides is sterically inhibited by the
anti-arylsulfatase A mAbs. To differentiate between these
possibilities, we developed an assay in which arylsulfatase A inhibits
the phosphorylation of arylsulfatase B competitively. If arylsulfatase
A-mAb complexes can still bind to the phosphotransferase, they should
be able to compete arylsulfatase B phosphorylation. If the mAbs prevent
arylsulfatase A from interacting with the phosphotransferase,
arylsulfatase A-mAb complexes should not compete arylsulfatase B phosphorylation.
Fig. 2 shows results of three independent
phosphorylation experiments. In these experiments arylsulfatase B and
arylsulfatase A were co-phosphorylated in the same reaction.
Arylsulfatase A was added in about 10-fold molar excess over
arylsulfatase B. Addition of arylsulfatase A inhibits arylsulfatase B
phosphorylation by about 40%. In all cases addition of
anti-arylsulfatase A Fab fragments abolishes the inhibitory effect of
arylsulfatase A on arylsulfatase B phosphorylation. Thus, Fab fragments
of all mAbs prevent interaction of arylsulfatase A with the
phosphotransferase.
Lysines Are Important for Recognition of Arylsulfatase A and
Arylsulfatase B by the Phosphotransferase--
The importance of
lysines for lysosomal enzyme recognition has been documented for
several lysosomal enzymes (6, 10-12). In order to investigate the role
of lysines in sulfatases, purified arylsulfatase A and
arylsulfatase B were treated with EDC and SNA to modify
glutamate/aspartate carboxylic groups and amino groups of
lysines, respectively (Fig. 3). EDC in
combination with ethylenediamine converts carboxylic groups
of glutamate and aspartate to the corresponding amides, SNA causes
acetylation of amino groups of lysines.
Modification of carboxyl groups affects arylsulfatase A and
arylsulfatase B in vitro phosphorylation only slightly
causing a reduction of phosphorylation by about 30-40% (see Fig. 3).
In contrast, lysine modification inhibited phosphorylation of
arylsulfatase A and arylsulfatase B completely. In case of modification
of carboxylic groups, the enzyme activity of arylsulfatase A was lost,
whereas acetylation of lysines left enzyme activity unchanged (data not shown). This excludes that an unspecific denaturing effect of the
modifications accounts for the loss of phosphorylation. Thus, lysines
are essential for the recognition of arylsulfatase A and arylsulfatase
B by the lysosomal enzyme phosphotransferase.
Effect of the Monoclonal Antibodies on pH-dependent
Octamerization of Arylsulfatase A--
The fact that all mAbs
inhibited the interaction of arylsulfatase A with the lysosomal enzyme
phosphotransferase raises the question of specificity. The mAbs may
interfere unspecifically with any protein-protein interactions of
arylsulfatase A simply by size. To get a measure of the specificity of
mAb inhibition of phosphorylation we have investigated the influence of
the mAbs on another protein-protein interaction of arylsulfatase A that can be performed in vitro. This is the
pH-dependent octamerization of arylsulfatase A. At neutral
pH, arylsulfatase A exists as a dimer, which upon acidification
associates to form octamers (23). To examine whether the mAbs interfere
with octamerization, 100 ng of purified arylsulfatase A was adsorbed to
a 96-well microtiter plate. 35S-Labeled arylsulfatase A was
added at neutral pH to the wells. Afterward, pH in the wells was
titrated to pH 4.5 under continuous shaking. At low pH, some of the
fluid phase 35S-labeled arylsulfatase A should associate
with arylsulfatase A bound to the solid phase via interaction of the
octamerization domain. If wells are washed at pH 4.5, labeled
arylsulfatase A remains bound to the solid phase (bars Co pH
4.5 in Fig. 4) whereas washing at
neutral pH reverses octamerization and removes most of the bound
[35S]arylsulfatase A (bars Co pH 7.4 in Fig.
4). To investigate whether mAbs interfere with this
pH-dependent octamerization process, mAbs were added either
to the wells to bind to solid phase arylsulfatase A before labeled
fluid phase [35S]arylsulfatase A was added (light
bars in Fig. 4) or mAbs were preincubated with fluid phase
[35S]arylsulfatase A before addition to wells (dark
bars, Fig. 4). After titration to pH 4.5 and shaking for 1 h,
wells were washed at pH 4.5 and counts remaining in the wells were
determined. Compared with control mAb, none of the arylsulfatase
A-specific mAbs inhibited binding of fluid phase
[35S]arylsulfatase A to solid phase enzyme, irrespective
whether mAbs were incubated with the fluid phase
[35S]arylsulfatase A or unlabeled arylsulfatase A bound
to the wells. Radioactivity in all wells could be removed by subsequent
washing at neutral pH, which reverses octamerization (data not shown). Control experiments showing that arylsulfatase A-mAb complexes are
stable at pH 4.5 have been performed (data not shown).
Simultaneous Binding of Monoclonal Antibodies to Arylsulfatase
A--
To evaluate how many different epitopes are recognized by the
mAbs, we investigated which of the mAbs can bind simultaneously to
arylsulfatase A. Wells of a 96-well microtiter plate were precoated with the eight anti-arylsulfatase A mAbs and with a control antibody. Arylsulfatase A was preincubated with a 10-fold molar excess of antibodies for 16 h and then added to the wells containing the immobilized mAbs. In case arylsulfatase A-bound mAbs compete binding of
the immobilized mAbs arylsulfatase A should not bind to the immobilized
mAbs in the wells. After 3 h incubation of arylsulfatase A-mAb
complexes in the wells, wells were washed and arylsulfatase A remaining
in the wells was quantified by activity determination. Results are
shown in Table I.
Antibodies A1, A2, A3, A4, and A5 compete when binding to arylsulfatase
A, which allows the conclusion that they recognize the same or adjacent
epitopes. Antibody C partially competes binding of E, but not of F, and
E also competes partially with F. It seems that these antibodies
recognize different epitopes. We have performed similar experiments
with two mAbs (B1 and B2) we have used in previously published
experiments (18). (For alterations in mAb designations see
"Experimental procedures.") These antibodies compete with the first
group of antibodies, but not with C, E, or F. This demonstrates that
the mAbs recognize at least four different epitopes and that at least
two mAbs can bind simultaneously to arylsulfatase A.
Epitopes of Anti-arylsulfatase A Monoclonal Antibodies--
To
localize epitopes, we immunoprecipitated human/murine arylsulfatase A
chimeric enzymes, compared mouse (24), rat and human cDNA predicted
amino acid sequences (25), and used information available from
crystallographic data of arylsulfatase A (23).
We have constructed chimeric arylsulfatase A enzymes, by fusing murine
and human arylsulfatase A cDNA sequences. These chimeric cDNAs
were expressed in baby hamster kidney cells and arylsulfatase A was
immunoprecipitated from the extracts. Arylsulfatase A activity was
measured in the supernatants and pellets after immunoprecipitation. Since the mAbs do not recognize the murine enzyme, this approach allows
to map the regions to which the mAbs bind. Table
II summarizes the results, according to
which the mAbs can be divided into four groups based on the different
patterns of chimeric arylsulfatase A immunoprecipitation. Since the
murine and rat enzyme are not recognized by the antibodies, comparison
of rat and murine cDNA predicted amino acid sequences with those of
the human cDNA allowed to locate epitopes further to differences in
the predicted amino acid sequences. Since the mAbs recognize the native
enzyme only, they should react with amino acid residues present on the
surface of the enzyme. Surface located residues can be inferred from
the three-dimensional structure of the arylsulfatase A enzyme, which we
have determined recently (23). Data from the chimeric enzymes, amino
acid sequence comparisons, and the criterion of surface location allow
the conclusion that mAbs A1, A2, A3, A4, and A5 bind to an epitope
between amino acids 18 and 97. MAb C binds to amino acids 165-169,
202-205, 216, or 239 and 240. Epitopes of a mAbs E and F can be less
precisely defined, because these mAbs bind to regions in which the
number of amino acids, which are different among mouse/rat and human
arylsulfatase A, is much higher than in regions to which the other mAbs
map. E binds between amino acids 287 and 459 and at least part of the
epitope of F is located between amino acids 463 and 507. MAbs D, B1,
and B2 have been described previously (18). The latter two show the same chimeric enzyme immunoprecipitation pattern as C, whereas the
epitope of D has been mapped to amino acids 256 to 260 (see Table
III for summary).
We have generated eight anti-human arylsulfatase A mAbs to study
the interaction of this lysosomal enzyme with the lysosomal enzyme phosphotransferase.
Results are based on an in vitro system in which mannose
6-phosphate residues as specific lysosomal targeting signals are synthesized in vitro on oligosaccharide side chains of
recombinant arylsulfatase A. All 8 anti-arylsulfatase A mAbs or their
Fab fragments, respectively, were able to inhibit in vitro
phosphorylation of arylsulfatase A completely. This inhibition may have
three different explanations. If all mAbs bind close to the lysosomal enzyme recognition determinant, it can be expected that they prevent interaction of arylsulfatase A with the phosphotransferase.
Alternatively, the interacting surface of arylsulfatase A with
phosphotransferase may be so large that any antibody will interfere
with recognition irrespective of the distance of its epitope to the
recognition determinant. Third, Fab fragments may bind to areas outside
the recognition site, such that arylsulfatase A-Fab fragment complexes may still be recognized by the phosphotransferase, but due to sterical
hindrance phosphate cannot be transferred to the oligosaccharide side chains.
The latter possibility has been excluded by competition experiments.
Arylsulfatase A-Fab fragment complexes do not inhibit arylsulfatase B
phosphorylation. This allows the conclusion that the Fab fragment
complexed arylsulfatase A does not interact with the
phosphotransferase. If the effect of the Fab fragments would be limited
to sterical hindrance of oligosaccharide phosphorylation, binding to
the phosphotransferase via the recognition determinant should have been
possible and arylsulfatase A-Fab fragment complexes should inhibit
arylsulfatase B phosphorylation.
To distinguish between the remaining two possibilities, we attempted to
locate the epitopes more precisely. Data from immunoprecipitation experiments of human/murine chimeric arylsulfatase A and mAb
competition experiments suggest that the mAbs, described here and
previously (18), recognize at least 6 different epitopes, all of which depend on the native status of the enzyme (see Tables II and III). Table III summarizes the location of the epitopes of anti-arylsulfatase A mAbs and lists possible amino acids being part of these epitopes. Data from these experiments are consistent with those from competition experiments, in which different mAbs were tested for their capability to bind arylsulfatase A simultaneously. MAbs mapping to the same region
by chimera immunoprecipitation also compete when binding to
arylsulfatase A. Vice versa, mAbs C, E, and F, which map to different
parts than A1 to A5 do not compete binding of these mAbs. Similarly,
the different epitope mapping pattern of mAbs C, E, and F as revealed
by immunoprecipitation of chimeric enzyme is also reflected in
competition experiments. C and F do not compete, but at least partial
competition can be seen between C/E and E/F. On the other hand,
competition experiments also show that antibodies mapping to the same
regions according to chimeric arylsulfatase A immunoprecipitation must
recognize different epitopes. MAbs B1, B2, and C show the same
reactivity pattern toward chimeric arylsulfatase A enzymes (Table II
and 3), but B1 and B2 do not compete binding of C proving recognition
of different epitopes.
The combination of all data allows location of likely positions of
epitopes in the three-dimensional model of arylsulfatase A as presented
in Fig. 5. Amino acids determining the
epitope of mAbs A1 to A5 (see Table III) are located on one side of the enzyme. Amino acids of B1/B2 and C are found on top and on the opposite
side of arylsulfatase A. Antibodies B1 and B2 do not compete with C,
although chimeric immunoprecipitation locates them to the same amino
acid residues. This is understandable in view of the three-dimensional
model: the residues determining the epitopes of these mAbs are found in
three distant locations so that simultaneous binding of these
antibodies is conceivable. Epitope of D has been determined previously
(18) and is found on the same side as those of B1, B2, and C. Epitopes
of mAbs E and F can be less precisely defined. However, their epitopes
must be located in some distance to those of the other antibodies, because they do not, or in the case of mAb E only partially, compete binding of these antibodies to arylsulfatase A. Thus, as illustrated in
Fig. 5 epitopes of mAbs are found on different parts of the surface of
arylsulfatase A. Therefore, the inhibition of phosphorylation cannot be
explained by binding of all antibodies to a restricted area on the
surface of arylsulfatase A.
INTRODUCTION
Top
Abstract
Introduction
References
-hairpin structure may be common to several
lysosomal enzymes (15). Whether this sequence is indeed part of the
recognition determinant common to all lysosomal enzymes remains to be proven.
EXPERIMENTAL PROCEDURES
-32P]ATP (3000 Ci/mmol)
was from Amersham. Enzymes used in the synthesis of
[
-32P]UDP-N-acetylglucosamine were
purchased from Sigma. All other chemicals were of analytical or
molecular biology grade and were from Merck, Serva, Sigma, or Fluka.
MAbs 5C6 (19) and NQ2/16-2 (20) were used as controls and were provided
by Dr. Lemke, Kiel, Germany and Dr. Gordon, Oxford, United Kingdom.
-light chains.
-32P]UDP-GlcNAc and arylsulfatase A
by immunoaffinity chromatography has been described in detail (18, 22).
Purified arylsulfatase B was kindly provided by M. Evers and C. Peters,
Freiburg, Germany. Quantification of radioactivity after SDS-PAGE was
done with a Fuji Bioimager.
RESULTS
cells overexpressing the human arylsulfatase A
cDNA. Since the sum of enzyme activity remaining in the supernatant
(in all cases less than 10%) and bound to the mAbs in the pellet was
in the same range as the activity in the supernatant when a irrelevant
control antibody was used, it can be concluded that the antibodies do
not interfere with arylsulfatase A activity (data not shown). All mAbs
recognize human arylsulfatase A specifically, they do not precipitate
rat or murine arylsulfatase A (less than 10% in all cases). They do not cross-react with arylsulfatase B an enzyme highly homologous to
arylsulfatase A. None of the mAbs recognizes arylsulfatase A under
denaturing conditions as revealed by Western blot analysis (data not shown).
-32P]UDP-GlcNAc from which the radioactively
labeled 32P is transferred to the oligosaccharide side
chains of the substrate enzymes. Subsequently, phosphorylation is
detected by SDS-PAGE analysis and autoradiography.
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Fig. 1.
Anti-arylsulfatase A monoclonal antibodies
inhibit the in vitro phosphorylation of arylsulfatase
A. Arylsulfatase A (ASA) and arylsulfatase B
(ASB) (0.5 µg each) were incubated in the presence of 8 µg of Golgi membranes and 22 µM
[ -32P]UDP-GlcNAc. After 2 h of incubation samples
were separated on a SDS-PAGE and subjected to autoradiography. Where
indicated, a 5-fold molar excess of anti-arylsulfatase A mAbs
(panel A) or Fab fragments (panel B) were added.
Co designates a control antibody, and
, no addition. The
addition of a 5-fold molar excess of anti-arylsulfatase A mAbs or Fab
fragments causes inhibition of arylsulfatase A phosphorylation, whereas
that of arylsulfatase B remains unaffected. As indicated, the
preparation of arylsulfatase B contains both the arylsulfatase B
precursor and the processed mature arylsulfatase B enzyme.
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Fig. 2.
Effect of anti-arylsulfatase A Fab fragments
on the inhibition of arylsulfatase B phosphorylation by arylsulfatase
A. Arylsulfatase A (ASA) (10 µg) and arylsulfatase B
(ASB) (1 µg) were phosphorylated in the presence of 0.75 µg of Golgi membranes. Anti-arylsulfatase A Fab fragments or Fab
fragments of a control mAb (Co) were added in 3-fold molar
excess. After 2 h of incubation samples were separated by
SDS-PAGE, and arylsulfatase B phosphorylation was determined by
quantification of radioactivity with a Fuji Bioimager. Additions of
arylsulfatase A or arylsulfatase B are indicated at the bottom. 100%
is the extent of arylsulfatase B phosphorylation that was obtained in
the sample containing the control Fab fragment. Addition of an excess
of arylsulfatase A inhibits arylsulfatase B phosphorylation by about
40%. Simultaneous addition of arylsulfatase A mAb Fab fragments
abolishes the inhibitory effect of arylsulfatase A on arylsulfatase B
phosphorylation. The columns indicate the mean of three
independent experiments. Vertical lines indicate the minimal
and maximal values of these experiments.
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Fig. 3.
In vitro phosphorylation of chemically
modified arylsulfatase A and arylsulfatase B. Arylsulfatase A
(ASA) (left panel) or arylsulfatase B
(ASB) (right panel) were treated with EDC to
modify carboxyl groups (Glu/Asp) or with SNA to modify amino groups
(Lys). + and indicate the treated and untreated samples,
respectively. In case of carboxyl modification the activity of the
enzymes was lost, it was entirely retained in the amino group modified
samples. The numbers in the bottom line give the
percentage of in vitro phosphorylation, where values of
unmodified samples are taken as 100%.
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Fig. 4.
Effect of anti-arylsulfatase A monoclonal
antibodies on the pH-dependent octamerization of
arylsulfatase A. 100 ng of arylsulfatase A was coupled to each
well of a 96-well microtiter plate. 35S-Labeled
arylsulfatase A was added to the wells after preincubation with a
10-fold molar excess of antibody indicated at the bottom
(dark bars). In a different experiment, antibodies were
added to the arylsulfatase A containing wells and
35S-labeled arylsulfatase A was added after the unbound
antibodies have been removed (light bars). After addition of
35S-labeled arylsulfatase A the pH in the wells was
titrated to 4.5 and plates were incubated for 1 h. Plates were
washed twice at pH 4.5 to remove unbound arylsulfatase A. Bars represent the radioactivity remaining in the wells.
Controls pH 4.5 and pH 7.4 were done without the addition of antibody.
Control pH 7.4 was washed at pH 7.4. At this pH octamerization does not
occur, and radioactive arylsulfatase A is removed. This represents the
background value. Control pH 4.5 was washed at acidic pH and represents
the 100% value for octamerization. Absolute values for control pH 7.4 are ~200 and ~800 cpm for control pH 4.5.
Competition assay of monoclonal antibody binding to human arylsulfatase
A
Epitope mapping by immunoprecipitation of chimeric enzymes
that less
than 10% of the enzyme was precipitable, and (+) indicates a 50%
reduction in immunoprecipitation. Part of the data on antibodies D, B1,
and B2 shown at the bottom have been published previously (18).
Summary of possible epitopes of anti-arylsulfatase A monoclonal
antibodies
DISCUSSION
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Fig. 5.
Location of epitopes in the three-dimensional
model of arylsulfatase A. The figure shows different views of a
three-dimensional model of arylsulfatase A. Coordinates according to
Brookhaven Protein Data Bank accession number PDB Id: 1AUK.
A and B, surface-located lysines are shown in
red, residues involved in octamerization in
green. Possible epitopes of mAbs A1 to A5 are depicted in
blue, those of B1, B2 and C mAbs are yellow, that
of D, gray. Asparagines bearing N-linked
oligosaccharide side chains are shown in black or
pink. They are labeled Ol1, Ol2, and Ol3 indicating
oligosaccharide side chains linked to asparagines 158, 184, and 350, respectively. Ol2 (pink) is not phosphorylated by the
lysosomal enzyme phosphotransferase (17). B shows a view of
the enzyme after vertical rotation by 180° compared with
A. C and D, lysine/octamerization area
and oligosaccharide labeling as in A and B.
Possible epitopes of mAb E appear dark purple, those of F,
tan. Epitopes can be less clearly defined because numerous
amino acid residues are different among mouse/rat and human
arylsulfatase A in the respective regions. D shows a view of
the enzyme after vertical rotation by 180° compared with
C.
Modification of lysines specifically inhibits the recognition of arylsulfatase A and arylsulfatase B by the phosphotransferase. In arylsulfatase A only five lysines are clustered on the surface of enzyme(23). These five lysines are not evenly distributed on the enzyme surface, but are located in a single cluster (see Fig. 5). Interestingly, this lysine cluster is in close proximity and partly overlaps the octamerization determinant of arylsulfatase A. The mAbs (except possibly for E and F) have epitopes that are distant from this region, which is functionally supported by the fact that none of the mAbs interferes with octamerization. Thus, there is no evidence that any of the mAbs or Fab fragments bind close to the lysine containing lysosomal enzyme recognition determinant in arylsulfatase A, yet all are able to inhibit binding of arylsulfatase A to the phosphotransferase. The fact that the mAbs do not inhibit octamerization, but phosphorylation suggests that the area interacting with the phosphotransferase is larger than the octamerization area, which covers about 900 Å (23) and thus larger than the lysine cluster necessary for proper recognition.
One may argue that the size relation of arylsulfatase A to Fab fragments (62 versus ~50 kDa) is unfavorable, so that an unspecific inhibition of protein-protein interactions can be expected. However, this seems to be unlikely, since Fab fragments have been shown to specifically inhibit functions of protein domains, which are limited to a few amino acids. Microinjections of Fab fragments generated against pentapeptides contained in the cytoplasmic tail of the 46-kDa mannose 6-phosphate receptor have shown that Fab fragments binding to amino acids 43-48 interfere with proper sorting of the receptor, whereas Fab fragments directed against amino acids 38-43 do not (26). This demonstrates that Fab fragments despite of their size can be used to discern protein-protein interactions even of adjacent small amino acid sequences. Co-crystallization of lysozyme and lysozyme-binding Fab fragments revealed that the binding site of the Fab fragment is a flat structure and the fragment extends from the binding site into the surrounding solution in a rather stretched conformation (27). The Fab fragment leaves most of the surface of the antigen accessible. The size of the interacting area of lysozyme and lysozyme Fab-fragments is 600 Å2. This is only a fraction of the total arylsulfatase A surface area. Thus, the ability of the mAbs to inhibit interaction with the phosphotransferase cannot be explained by the assumption that single Fab fragments cover large parts of the arylsulfatase A surface making them inaccessible to other molecules. This is supported by our demonstration that none of the mAbs interferes with enzyme activity or octamerization and that at least two mAbs can bind simultaneously to the enzyme.
Our data support a model in which lysosomal enzyme phosphotransferase interacts with large surface areas of arylsulfatase A, a view that can be brought into line with results of targeted amino acid substitutions in cathepsin D (10, 12) and aspartylglucosaminidase (11). In cathepsin D two lysine residues 34 Å apart (203 and 293) cooperate to form part of a phosphotransferase recognition determinant. When these residues are mutated 30% of normal phosphorylation still remains (12), demonstrating the involvement of more residues and thus, most likely a larger surface area in the formation of a fully efficient recognition determinant. In aspartylglucosaminidase three distantly located lysine residues contribute to form a recognition signal. In addition, the minimal regions establishing the cathepsin D recognition determinants were identified as lysine 203 and amino acid residues 265-292. When these residues were introduced into the non-lysosomal homologous protease glycopepsinogen, the chimeric molecule was recognized by the phosphotransferase, but to a much lesser extent than cathepsin D (6). Obviously, residues beyond lysine 203 and amino acids 265-292 contribute to efficient expression of the lysosomal enzyme recognition determinant, and it has been shown that such sequences can be found in the amino-terminal lobe of cathepsin D (8, 10). A minimal recognition sequence may be necessary for recognition, but its efficiency can be enhanced by additional even distant areas on the same molecule. The cooperativity of various surface areas in the formation of a fully expressed lysosomal enzyme recognition determinant can be explained by different models in which the distant parts of a lysosomal enzyme bind subsequently or simultaneously to phosphotransferase (10). Phosphotransferase may initially bind lysosomal enzymes through a lysine-dependent recognition determinant and starts to phosphorylate accessible oligosaccharide side chains. After completion of this phosphorylation step, the enzyme dissociates and rotates to get access to another oligosaccharide side chain. However, the lysosomal enzyme remains in the vicinity of the phosphotransferase ensuring a high probability of rebinding even at weaker lysine-dependent or independent sites, present at different locations on the lysosomal enzyme surface. This process may continue until several oligosaccharide side chains have been phosphorylated. If this model is correct, it is surprising that none of the arylsulfatase A-Fab fragment complexes inhibits the phosphorylation of arylsulfatase B. Since the lysine region on arylsulfatase A is not covered by any of the Fab fragments, an initial contact through this region should not be disturbed by Fab fragments and arylsulfatase A-Fab fragment complexes should be able to inhibit phosphorylation of arylsulfatase B competitively. Thus, a model which depends on an initial high affinity binding of restricted area and with subsequent rotation and rebinding at weaker sites seems not likely.
Alternatively, phosphotransferase may contact the surface of lysosomal
enzyme at multiple sites simultaneously. The initial contact may depend
on a lysine containing recognition determinant and subsequently binding
is stabilized via additional contacts. Since phosphotransferase is a
large multimeric enzyme of 9 times the molecular weight of an
arylsulfatase A monomer, such an explanation seems applicable. Since
all Fab fragments irrespective of their binding sites prevent
interaction of arylsulfatase A with the phosphotransferase, it may be
assumed that lysosomal enzymes enter a large active site pocket
covering most of lysosomal enzymes surface. However, since lysosomal
enzymes have different shapes it seems unlikely that they all fit
tightly into a large inflexible active site of phosphotransferase.
Thus, after initial contacts are made perhaps by
charge-dependent interactions with lysines a conformational
change in the phosphotransferase may be induced which causes
embracement of the lysosomal enzyme. This embracement is only
stabilized when additional contact points are established, which may
then occur at distant points on the surface of a lysosomal enzyme. In
this case the arylsulfatase A-Fab fragment complexes may still interact
with phosphotransferase but Fab fragments may interfere sterically with
embracement and establishment of secondary stabilizing contacts. Since
arylsulfatase B phosphorylation cannot be inhibited by arylsulfatase
A-Fab fragment complexes, in the embracement model the initial lysine
dependent contact must be of low affinity. Our data does not allow
distinction between the latter models, but in any case support a model
in which large areas of the surface of lysosomal enzymes simultaneously
interact with the phosphotransferase.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Evers and Dr. C. Peters for providing purified arylsulfatase B. We also thank Dr. H. Lemke for critical comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Gi 155/6-1.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.
The atomic coordinates and structure factors (code 1AUK) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed: Biochemisches Institut
der Christian-Albrechts-Universität, Olshausenstrasse 40, 24098 Kiel, Germany. Tel.: 49-431-880-2212; Fax: 49-431-880-2007; E-mail:
office{at}biochem.uni-kiel.de.
The abbreviations used are: phosphotransferase, UDP-N-acetylglucosamine:lysosomal enzyme N-acetyl-glucosamine-1-phosphotransferase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; SNA, sulfo-N-hydroxy-succinimidylacetate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
2 F. Dietz, unpublished data.
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REFERENCES |
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