Results wtASA activity is diminished upon coexpression with misfolded ASA polypeptides In a number of experiments in which we expressed misfolded, enzymatically inactive ASA to investiga
Trang 1degradation of wild-type subunits of arylsulfatase A
heteromers
Peter Poeppel1,*, Mekky Mohamed Abouzied1,2, Christof Vo¨lker1 and Volkmar Gieselmann1
1 Institut fu¨r Biochemie und Molekularbiologie, Rheinische-Friedrich-Wilhelms Universita¨t Bonn, Germany
2 Faculty of Pharmacy, University of El-Minia, Egypt
Introduction
Many proteins form homooligomers According to
crystallization and in vitro gel filtration data, the
lyso-somal enzyme arylsulfatase A (ASA; UniProt accession
number P15289) forms dimers at neutral pH and
octa-mers at acidic pH [1,2] ASA is a 62 kDa soluble
protein with three N-linked oligosaccharide side chains
[3] Within the Golgi apparatus, mannose residues of
at least two of these side chains are phosphorylated
The resulting mannose 6-phosphate residues are
impor-tant for mannose 6-phosphate receptor-mediated
lyso-somal delivery of the enzyme A polymorphism that is
frequent in the normal population (allele frequency of approximately 15%) causes substitution of asparagine
350 carrying the third N-linked oligosaccharide of the enzyme by serine This substitution abolishes the N-glycosylation site Therefore, this allele codes for a slightly smaller ASA with only two oligosaccharide side chains [4] This ASA has been termed pseudodefi-ciency ASA (pdASA) Despite the loss of one N-linked oligosaccharide side chain, the biochemical properties
of the pdASA polypeptide are largely identical to those
of the wild-type ASA (wtASA) [4,5]
Keywords
arylsulfatase A; ERAD; MLD; protein
oligomerization; protein quality control
Correspondence
V Gieselmann, Institut fu¨r Biochemie und
Molekularbiologie,
Rheinische-Friedrich-Wilhelms Universita¨t Bonn, Nussallee 11,
53115 Bonn, Germany
Fax: +49 228 732416
Tel: +49 228 732411
E-mail: gieselmann@ibmb.uni-bonn.de
*Present address
Institut fu¨r Biochemie, Universita¨t zu Ko¨ln,
Germany
(Received 10 February 2010, revised 6 May
2010, accepted 18 June 2010)
doi:10.1111/j.1742-4658.2010.07745.x
Arylsulfatase A is an oligomeric lysosomal enzyme In the present study,
we use this enzyme as a model protein to examine how heteromerization of wild-type and misfolded endoplasmic reticulum-degraded arylsulfatase A polypeptides affects the quality control of wild-type arylsulfatase A subun-its Using a conformation sensitive monoclonal antibody, we show that, within heteromers of misfolded and wild-type arylsulfatase A, the wild-type subunits are not fully folded The results obtained show that arylsulfatase
A polypeptide complexes, rather than the monomers, are subject to endo-plasmic reticulum quality control and that, within a heteromer, the mis-folded subunit exerts a dominant negative effect on the wild-type subunit Although it has been shown that mature lysosomal arylsulfatase A forms dimers at neutral pH, the results obtained in the present study demonstrate that, in the early biosynthetic pathway, arylsulfatase A forms oligomers with more than two subunits
Abbreviations
ASA, arylsulfatase A; ER, endoplasmic reticulum; ERAD, ER associated degradation; HA, hemagglutinin tag; MLD, metachromatic
leukodystrophy; moab, monoclonal antibody; pdASA, pseudodeficiency arylsulfatase A; UGGT, UDP-glucose:glycoprotein glucosyltransferase; wtASA, wild-type arylsulfatase A.
Trang 2Deficiency of ASA causes metachromatic
leukodys-trophy a lysosomal storage disorder, in which the
degra-dation of the sphingolipid 3-O sulfogalactosylceramide
is interrupted [6] This leads to progressive
demyelina-tion and finally lethal neurologic symptoms ASA
deficiency is frequently caused by missense mutations,
which cause misfolding and endoplasmic
reticulum-associated degradation (ERAD) of the respective ASAs
[7] Because the pd allele is so frequent, a number of
mutations causing metachromatic leukodystrophy
(MLD) were identified, which occur on the background
of this allele One of these missense mutations causes a
P377L substitution, leading to ERAD of the respective
ASA [8]
ERAD occurs when a protein does not pass the
ER quality control [9] Various degradation pathways
exist in the ER The best characterized of these
path-ways is proteasomal degradation of glycoproteins,
which involves the modification of their N-linked
oligosaccharide side chains [10] These Glc3Man9
Glc-NAc2 oligosaccharide side chains are processed to a
Glc1Man9GlcNAc2 structure by ER glucosidases I
and II This oligosaccharide side chain allows binding
to calnexin or calreticulin ER resident chaperones
Deglucosylation of the Glc1Man9GlcNAc2 structure
by ER glucosidase II releases glycoproteins from
calnexin⁄ calreticulin [10] UDP-glucose:glycoprotein
glucosyltransferase (UGGT) functions as a folding
sensor and reglucosylates the Man9GlcNAc2
oligosac-charides in case the released newly-synthesized
protein is not properly folded This leads to
reassociation with calnexin⁄ calreticulin and a new
cycle to achieve correct folding If the protein does
not succeed in achieving the correct conformation
after repetitive deglucosylation⁄ reglucosylation cycles,
it will be targeted for proteasomal degradation
[11,12]
It is largely unclear how UGGT recognizes
specifi-cally misfolded proteins In vitro experiments using a
heterodimer of normal and misfolded RNAse B [11]
demonstrated that the enzyme recognizes and
reglu-cosylates selectively the misfolded subunit of the
hete-rodimer Although oligomerization of proteins is a
frequent phenomenon, little is known with respect to
how ER quality control deals with heterooligomers of
wild-type and misfolded proteins The variety of
defective ASAs, which are subject to ERAD [7], and
the availability of structure-sensitive monoclonal
anti-bodies identify ASA as a model protein well suited
for an investigation of the consequences of
hetero-merization of wild-type and defective proteins in more
detail
Results wtASA activity is diminished upon coexpression with misfolded ASA polypeptides
In a number of experiments in which we expressed misfolded, enzymatically inactive ASA to investigate the biochemical consequences of missense mutations,
we noted that this reduced the endogenous ASA activ-ity of the transfected cells To examine this phenome-non in more detail, we coexpressed active wild-type and defective enzymes and measured ASA activity in cell lysates (Fig 1) Two aspects must be considered in the set up of this experiment:
The first is the fraction of cells coexpressing wild-type and defective ASA after transient transfection To determine the percentage of cells coexpressing both types of enzyme, we transiently transfected BHK cells with various amounts of plasmid expressing either green fluorescent protein or DsRed fluorescent proteins After transfection, cells expressing both proteins were counted using an immunofluorescence microscope Independent of the amount of DNA transfected, 70–73%
of cells expressed both proteins (data not shown) The second fact for consideration is that sulfatases bear a unique modification of a cysteine residue in the active center [13] Cotranslationally, this residue is con-verted to formylglycine, which is essential for enzyme activity [13] Overexpression of sulfatases can saturate the formylglycine-generating enzyme, so that a fraction
of the newly-synthesized sulfatases remains inactive Therefore, the easiest explanation for a reduction of wtASA activity upon coexpression of a defective enzyme
is that the latter competitively displaces the wild-type enzyme from the formylglycine-generating enzyme [14,15] To exclude this effect, we transiently transfected increasing amounts of a plasmid expressing wtASA and measured enzyme activity in cells Figure 1A shows that the correlation between the transfected amount of wtASA expressing plasmid and ASA activity is approxi-mately linear in the range 0–10 ng of plasmid Transfec-tion of more than 10 ng of plasmid does not lead to a substantial further increase of ASA activity By contrast, when the same cells were investigated by immunoprecipitation, the amount of ASA cross-react-ing material correlated with the amount of transfected plasmid up to 250 ng (data not shown) Thus, at higher plasmid concentrations, most of the synthesized ASA is inactive, most likely as a result of incomplete formylgly-cine residue formation In the range 0–10 ng of plasmid, however, the amount of ASA activity increases propor-tionally, showing that it is not limited by the activity of
Trang 3the formylglycine-generating enzyme We choose this
linear range and cotransfected 4.2 ng of plasmid
expressing wtASA cDNA with 4.2 ng of plasmid
expressing ASA cDNAs coding for various amino acid
substituted misfolded ASAs, which have been shown to
be enzymatically inactive and degraded by ERAD [7]
Coexpression of seven different defective, enzymatically
inactive enzymes in BHK cells caused a reduction of
activity to approximately 50% of controls (Fig 1B) The experiments were repeated in HEK293 cells, with identical results being obtained (data not shown)
Defective ASA causes partial retention of wtASA
To verify the results shown in Fig 1 by a different experimental approach, we performed pulse chase experiments in cells coexpressing wtASA and the defective P377L-pdASA We chose the P377L-pdASA because this is a missense mutation occurring on the background of the ASA pseudodeficiency allele As explained in the Introduction, pdASA is a naturally occurring variant lacking one of the three ASA oligo-saccharide side chains The properties of pdASA, how-ever, are largely identical to wtASA [8] Because of the loss of one N-linked oligosaccharide side chain, pdASA and P377L-pdASA have a lower apparent molecular weight by SDS⁄ PAGE and can be easily dif-ferentiated from wtASA (Fig 2A, bottom) WtASA and P377L-pdASA were expressed separately or together in BHK cells (Fig 2) Sixteen hours after transfection, cells were treated with NH4Cl This drug interferes with the post Golgi sorting of lysosomal enzymes and causes the secretion of newly-synthesized enzymes into the medium of cultured cells When cells were transfected with the wtASA cDNA, they were pulse labeled for 2 h and chased in the presence of
NH4Cl To quantify wtASA present in the medium at different chase times, we took the amount of wtASA present in the media and cells as 100% for each time point separately and plotted the percentage of ASA found in media against the chase time (Fig 2A, top) After 20 h of chase, the majority of wtASA is secreted
By contrast, the defective P377L-pdASA remains in the cells (Fig 2A, middle), which is expected as a result of the retention of the defective enzyme in the ER [8] In addition, the continuous reduction of the amount of defective enzyme during the chase period demonstrates its degradation When wtASA and P377L-pdASA were coexpressed, only the wild-type enzyme appeared in the medium, but not P377L-pdASA (Fig 2A, bottom) Quantification of the percentage of wtASA in the med-ium of NH4Cl-treated cells expressing either the wtASA alone or together with the P377L-pdASA revealed that the coexpression of the P377L-pdASA decreases the percentage of wtASA polypeptides in the medium to approximately half of the percentage found in cells expressing wtASA only (Fig 2B) This indicates that the defective P377L-pdASA is able to cause retention
of a fraction of wtASA in the cells
The quantification of total precipitated ASA (i.e sig-nals from cells plus medium for each chase time)
A
B
Fig 1 ASA activity after coexpression of wild-type and various
defective ASAs (A) Increasing amounts (0.5–40 ng) of a plasmid
encoding wtASA were transiently transfected into BHK cells and
enzyme activity was measured 48 h after transfection In the range
0.5–10 ng of plasmid, ASA activity increases in an almost
propor-tional manner (B) Some 4.2 ng of plasmid expressing wtASA was
cotransfected with 4.2 ng of plasmids expressing various inactive,
misfolded ASAs (P377L-pdASA, D335V-ASA, T275M-ASA,
P136L-ASA, G86D-P136L-ASA, T201C-ASA and D255H-ASA) In the control,
these plasmids were replaced by the empty vector (pBEH) The
activity value obtained at 40 ng was taken as 100% In all cases of
coexpression of defective ASA, the wtASA activity was lowered
below the level of expression of wild-type enzyme only ASA
activ-ity was determined as mUÆmg)1 protein When expressed alone,
none of the defective ASA polypeptides displays enzymatic activity
(data not shown).
Trang 4allows an investigation of whether coexpression of
P377L-pdASA with wtASA reduces the stability of the
latter Figure 2C shows that wtASA, after an initial
slight decrease, is quite stable, with 80% of the enzyme
still present after 20 h By contrast, only 20% of the
P377L-pdASA is left after 20 h Upon coexpression of
wild-type and defective enzyme, the amount of wtASA
after 20 h is reduced to less than 50% Obviously, the
defective P377L-pdASA enzyme leads to a more rapid
degradation of a fraction of the wild-type enzyme By
contrast, coexpression of the defective enzyme with
wild-type enzyme does not enhance the half-life of the
P377L-pdASA Thus, the defective enzyme has a
domi-nant negative effect on wtASA
Wild-type and misfolded ASA polypeptides form heteromers
The experiments presented in Figs 1 and 2 suggest an interaction of misfolded ASA and wtASA Because gel filtration and crystallization studies demonstrate that ASA forms oligomers [1,2], heteromerization of wild-type and defective ASA subunits may offer an explana-tion for the dominant negative effect observed To detect heteromerization of ASA in metabolic label-ing⁄ pulse experiments, wtASA was tagged with a nine amino acid hemagglutinin (HA) peptide sequence at the C-terminus to allow precipitation with monoclonal antibody specific for HA (HA moab)
Figure 3A shows that the HA moab immunoprecipi-tates HA tagged wtASA (wtASA-HA), but not untag-ged pdASA or P377L-pdASA Upon cotransfection, however, the pdASA P377L-pdASA coimmunoprecipi-tates with wtASA-HA, showing that this experimental set up allows the examination of ASA heteromeriza-tion In addition to wtASA, various defective ASAs were fused to the HA peptide sequence This yielded plasmids designated D335V-ASA-HA,
T274M-ASA-HA, P136L-ASA-T274M-ASA-HA, G86D-ASA-HA and D255H-ASA-HA All of the respective missense mutations
A
B
C
Fig 2 Secretion and stability of wtASA is decreased by the coex-pression of defective P377L-pdASA (A) BHK cells were transfected with plasmids conferring expression of wtASA or P377L-pdASA Cells expressed these enzymes alone (upper two panels) or in combination (lower panel) Cells were labeled with 370 kBq of [ 35 S]methionine for 2 h and chased for the times indicated in the presence of 10 m M NH4Cl ASA was immunoprecipitated from cell lysat (C) and media (M) and subjected to SDS ⁄ PAGE When cells were harvested immediately after the pulse period (left lane), ASA was only immunoprecipitated from the cells and not from the media With longer chase periods, increasing amounts of wtASA appear in the media (B) 35S-labeled wtASA polypeptides of two parallel experiments shown in (A) were quantified in the cells and the media The graph shows the percentage of wtASA present in the medium (for calculations, see text) Filled circles, cells only expressing wtASA; open circles, coexpression of defective P377L-pdASA The graph demonstrates that the coexpression of defective ASA reduces the secretion of wtASA Values represent the mean, minima and maxima of two parallel experiments Labeled polypep-tides were quantified using a Fuji bioimager (C) Graph showing the total amount of ASA polypeptides present in the cells and media at different chase times The amount of ASA polypeptides present after 2 h of pulse was taken as 100% Whereas wtASA (filled cir-cles) is stable over a time period of 20 h, P377L-pdASA (open squares) is rapidly degraded The half-life of wtASA is reduced upon coexpression of defective P377L-pdASA (open circles) The half-life of P377L-pdASA is unchanged upon coexpression of wtASA (closed squares) Values are the mean, minima and maxima
of two parallel experiments Labeled polypeptides were quantified
by Fuji bioimager.
Trang 5were described in MLD patients, shown to be retained
in the ER [16–18] and were degraded by the
protea-some [7] These defective HA tagged ASAs were
coex-pressed with pdASA in BHK cells (Fig 3B) Cells
were metabolically labeled with [35S]methionine for
30 min and HA tagged misfolded ASA polypeptides
were immunoprecipitated with the HA moab Resolution
of the immunoprecipitates by SDS⁄ PAGE revealed coimmunoprecipitation of untagged nondefective pdASA
in all HA immunoprecipitates of HA tagged defective ASAs Thus, pdASA forms heteromers with all of the misfolded ASAs examined
To exclude the possibility that heteromer formation
is not an in vivo phenomenon but occurs after cell lysis during immunoprecipitation, we labeled cells expressing either wtASA-HA or pdASA only After harvesting,
we mixed the cell lysates and performed immuno-precipiation with HA moab Under these conditions, wtASA-HA did not coimmunoprecipitate pdASA, demonstrating that heteromer formation does not occur during immunoprecipitation but in the cells (data not shown)
Stoichiometry of ASA oligomers
In the case where ASA forms dimers in the early stages
of biosynthesis, coexpression of equal amounts of wtASA-HA and pdASA will yield one-third wtASA-HA homodimers, one-third wtASA-HA⁄ pd ASA heterodi-mers and one-third pdASA homodiheterodi-mers This predicts that coimmunoprecipitation of the untagged pdASA
by the wtASA-HA should yield intensity ratios of the respective bands on SDS⁄ PAGE of approximately Transfection
Transfection
anti-hASA
anti-HA
anti-HA
Untagged
A
B
Fig 3 Detection of ASA heteromers A nine amino acid HA tag was added to the C-terminus of wtASA (wtASA-HA) or ASAs carry-ing various amino acid substitutions (D335V-ASA-HA,
T274M-ASA-HA, P136L-ASA-T274M-ASA-HA, G86D-ASA-HA and D255H-ASA-HA) (A) BHK cells were transiently transfected with the indicated expression vectors PdASA and P377L-pdASA only have two oligosaccharide side chains, resulting in a lower molecular weight compared to wtASA-HA Cells were labeled with 370 kBq of [35S]methionine for
2 h and subsequently harvested Cell lysates were divided into two aliquots and ASA polypeptides were immunoprecipitated with poly-clonal antiserum specific for ASA (upper panel) or HA moab (lower panel) Immunoprecipitates were resolved on SDS ⁄ PAGE and labeled polypeptides were visualized using a Fuji bioimager The polyclonal ASA antiserum precipitates all polypeptides, whereas the
HA moab precipitates only the HA tagged ASA polypeptides In cotransfected cells (lanes 4 and 5), untagged pdASA and P377L-pdASA are coimmunoprecipitated with the HA tagged wtASA-HA (lower panel) (B) Different HA tagged defective ASA polypeptides,
as indicated in the top line, were transiently expressed with pdASA, as indicated in the line below After transfection of BHK cells with equal amounts of vectors expressing the HA-tagged and untagged pdASAs, cells were labeled with 3.7 MBq [ 35 S]methio-nine for 30 min Subsequently, the cells were lysed and HA tagged ASAs were immunoprecipitated with an HA moab from cell lysates Immunoprecipitates were resolved on SDS ⁄ PAGE and labeled polypeptides were visualized using a Fuji bioimager pdASA coimmunoprecipitated with the various HA tagged defective ASA polypeptides.
Trang 6one-third (pdASA) and two-thirds (wtASA-HA),
respectively
Whereas this is the case as shown in Fig 3B, in
Fig 3A, the stoichiometry is not what was expected If
ASA was not present as a dimer but rather as an
oligomer in the early biosynthetic pathways,
differ-ences in the transfection efficiencies of the two
plas-mids encoding wtASA-HA and P377L-pdASA used in
Fig 3 could account for the variation in ratio of the
two associated ASA polypeptides For that reason, we
decided to examine the stoichiometry of the ASA
het-eromers in more detail by varying the ratio of the
amount of plasmids in a cotransfection experiment
We transfected BHK cells with varying amounts of
plasmid expressing wtASA-HA and pdASA or
P377L-pdASA, respectively Ratio of plasmids varied from
20% : 80% to 80% : 20%, respectively After
meta-bolic labeling, the cell lysates were split into two
aliqu-ots One aliquot was immunoprecipitated with a
polyclonal ASA antiserum precipitating all expressed
ASAs to control whether the ratios of wtASA and
pdASA or P377L-pdASA polypeptides really reflect
the ratios of the respective plasmids used for
transfec-tion Figure 4 shows that, except for minor deviations,
this is the case The second aliquot was
immunoprecip-itated with the HA moab to determine the amount of
the coimmunoprecipitated non-HA tagged pdASA
(Fig 4B) Quantification of the immunoprecipitated
wtASA-HA and coimmunoprecipitated pdASA or
P377L-pdASA, respectively, revealed that one
wtASA-HA coimmunoprecipitates at least five non-wtASA-HA tagged
pdASA polypeptides This suggests that
newly-synthe-sized ASA is present at least as a hexamer
Folding status of wtASA heteromerized with
mutant ASA
We have recently shown that wtASA folds in a
sequen-tial way, which can be followed by immunoprecipitation
with various structure-sensitive monoclonal antibodies
[7] The hASA specific moab A2 [19] detects an epitope
of wtASA that is already formed within the first few
minutes after biosynthesis [7] The T274M substituted
ASA, however, is severely misfolded, so that it does not
express this epitope and cannot be immunoprecipitated
by moab A2 [7] This prompted us to investigate
whether the T274M-ASA affects folding of the wtASA
occurring in the same heteromer Cells were transfected
with wtASA-HA, wtASA-Myc and T274M-ASA-HA,
respectively, or cells were cotransfected with different
amounts of wtASA-Myc and T274M-ASA-HA Cell
lysates were divided into three aliquots and the ASAs
were immunoprecipitated either with polyclonal ASA
antiserum or Myc epitope specific moab or with the hASA moab A2 The polyclonal antiserum is able to immunoprecipitate ASA even under denaturing conditions Immunoprecipitates were subjected to SDS⁄ PAGE followed by western blotting with the HA moab
When wtASA-HA was expressed and immunopre-cipitated with either polyclonal antiserum or the ASA moab A2, the HA moab detected wtASA-HA in the western blot of the immunoprecipitate This confirms that wtASA-HA is correctly folded and can therefore
be immunoprecipitated with moab A2 (Fig 5, lane 1) When T274M-ASA-HA was examined in the same way, no ASA polypeptides were detected with the HA moab after immunoprecipitation with the moab A2, confirming that incorrectly folded T274M-ASA-HA cannot be immunoprecipitated by the structure-sensi-tive ASA moab A2 (Fig 5, lane 3)
When wtASA-Myc was coexpressed with the T274M-ASA-HA and immunoprecipitated with the moab A2, again, no HA containing enzyme could be detected
in the immunoprecipitate (Fig 5, lane 4) This shows
A
B
Precipition anti-hASA
Precipition anti-hHA
Fig 4 Stoichiometry of ASA oligomerization To determine the stoichiometry of ASA in the oligomer, BHK cells were
cotransfect-ed with plamids expressing wtASA-HA and P377L-pdASA or pdASA, respectively The ratio of wtASA expressing plasmids to pdASA or P377L-pdASA expressing plasmids, respectively, varied,
as indicated at the top Cells were labeled with 4.1 MBq of [ 35 S]-methionine for 30 min After harvesting, cell lysates were split into two aliquots One aliquot was precipitated with polyclonal ASA anti-serum (A) and the other aliquot with the HA moab (B) Quantifica-tion of 35 S-labeled ASA polypeptides using a Fuji bioimager shows that one wtASA-HA can coimmunoprecipitate at least five untagged pdASA polypeptides.
Trang 7that the wild-type enzyme associated with the misfolded
T274M-ASA defective enzyme does not express the
A2 epitope and therefore is not completely folded
Otherwise, T274M-ASA-HA should be detectable in
the immunoprecipitate As a control demonstrating
wtASA-Myc and T274M-ASA-HA heteromerization, lysates from coexpressing cells were
immunoprecipitat-ed with the Myc moab Western blot analysis of these immunoprecipitates shows that the T274M-ASA-HA subunits are detectable by the HA moab after cotrans-fection with wtASA-Myc (Fig 5, middle) This reveals that the inability to coimmunoprecipitate the T274M-ASA-HA with the structure-sensitive moab A2 is not the result of a lack of heteromerization of T274M-ASA-HA and wtASA-Myc
These results suggest that, within a heteromer, the T274M substituted ASA prevents proper folding of the wtASA Improper conformation of the wtASA in the heteromer may be the result of the insufficient time available for folding because degradation as a result of association with the defective enzyme may occur too rapidly Kifunensine is an inhibitor of ER a1,2-man-nosidase I Inhibition of this enzyme blocks the path-way diverting a misfolded enzyme to the proteasome, allowing more time for proper folding [20] Therefore, wtASA and T274M-ASA-HA were coexpressed in the absence or presence of kifunensine ASA was immuno-precipitated with the moab A2 and the immunoprecipi-tates were probed on a western blot with the HA moab (data not shown) However, even after stabiliza-tion with kifunensine, T274M-ASA-HA could not be coimmunoprecipitated with wtASA, indicating that, under these conditions, wtASA expressing the epitope
of the moab A2 was not present in the
T274M-ASA-HA⁄ wtASA heteromers
Discussion Oligomerization of proteins is a frequent phenomenon, but the mechanism by which heterooligomers of normal and defective proteins pass ER quality control is only poorly understood We used the lysosomal enzyme ASA to examine the consequences of heteromerization
of wild-type and defective ASA in more detail
We demonstrate that, within such a heteromer, the misfolded ASA exerts a dominant effect on the wtASA subunit, decreasing its stability Although we have only shown this in detail for the P377L-pdASA, the reduc-tion of enzyme activity upon coexpression of various defective ASAs (Fig 1), as well as the capability of heteromerization for all defective ASAs investigated in the present study, strongly suggests that this applies to all defective ASA polypeptides
Crystallization [2] and gel filtration experiments [1] suggest that ASA forms dimers at neutral pH These studies were performed with mature lysosomal ASA, which has passed the biosynthetic compartments and reached its final lysosomal destination Our data,
Immuno-precipitation
Transfection
Western
Polyclonal
antiserum
anti-Myc
anti-HA
anti-HA
anti-HA Moab
A2
Fig 5 Association of misfolded T274M-ASA-HA with wtASA-Myc
prevents folding of wtASA-Myc BHK cells were transfected with
plasmids expressing either wtASA-HA (lane 1), wtASA-Myc (lane 2)
or T274M-ASA-HA alone (lane 3), or combinations of wtASA-Myc
and T274M-HA (lane 4), as indicated at the top ASA was
immuno-precipitated from cell lysates with either a polyclonal ASA
antise-rum recognizing ASA polypeptides even under denaturing
conditions (upper panel), or an Myc tag specific moab (middle
panel), or the structure sensitive hASA moab A2 (lower panel)
recognizing an epitope that is formed early in biosynthesis [7].
Immunoprecipitates were subjected to western blot analysis by HA
moab In the case of the immunoprecipitation with polyclonal
anti-serum, the HA tagged ASAs can be detected from cells expressing
wtASA-HA or T274M-ASA-HA alone and from cells coexpressing
20% wtASA-Myc and 80% T274M-ASA-HA (upper panel, lanes 1,
3 and 4) When Myc moab is used for immunoprecipitation,
T274M-ASA-HA can be detected by western blot analysis from
cells coexpressing 20% wtASA-Myc and 80% T274M-ASA-HA
(middle panel, lane 4) After immunoprecipitation with moab A2,
wtASA-HA can be immunoprecipitated from cells and detected by
western blotting (lower panel, lane 1) The defective
T274M-ASA-HA cannot be immunoprecipitated with the structure sensitive
moab A2 Also, in the case of coexpressing wtASA-Myc with
T274M-ASA-HA, the defective enzyme cannot be
coimmunoprecipi-tated with wtASA-Myc, indicating that wtASA-Myc does not
express the A2 epitope and thus is not completely folded.
Trang 8however, clearly show that, during the early
biosyn-thetic stages in vivo, the enzyme forms at least
hexa-mers, possibly octamers, which have only been
described in vitro at acidic pH The unexpectedly high
number of ASA monomers with an oligomer cannot
be explained by the aggregation of defective ASA
poly-peptides in the ER because the same stoichiometry is
also found with nondefective pd ASA Currently, we
do not have an explanation for the stoichiometry of
ASA, although it is possible that as yet unknown
mod-ifications occur during the early biosynthetic stages
that affect oligomerization of the enzyme
In vitro experiments using heterodimers of native
and misfolded RNAse B have demonstrated that, even
within a RNAse heterodimer, UGGT can distinguish
the native subunit from the misfolded subunit and
reglucosylates only the latter [11] If this process also
occurs similarly in vivo, the wtASA is expected to
remain unglucosylated within the ASA heteromers and
only the associated defective ASA would be
reglucosy-lated Because wtASA is also trapped in the ER, the
dominant effect of the defective enzyme may then be
explained by the assumption that a single misfolded
subunit causes degradation of the heteromer
irrespec-tive of the conformational status of the other subunits
The results obtained for the T274M-ASA, however,
offer yet another explanation The ASA moab A2 does
not recognize denatured ASA [19] Recent data suggest
that the antibody binds an epitope that is formed early
in ASA biosynthesis when the enzyme is partially
folded [7] The T274M substituted ASA does not react
with moab A2, nor with any other structure-sensitive
ASA moab, indicating that it is severely misfolded
This allowed an investigation of the folding status of
the wtASA within a heteromer with defective
T274M-ASA If the wtASA reaches a folding state within a
heteromer that allows the expression of the A2
epi-tope, it should be possible to immunoprecipitate the
wtASA with the moab A2 and to detect the
T274M-ASA-HA subunit afterwards in the
immunoprecipi-tates In our experiments, however, this was not the
case This suggests that, within the heteromer, the
wtASA does not fully proceed through its normal
fold-ing pathway Alternatively, the foldfold-ing of ASA may be
catalyzed by different chaperones acting successively
on the enzyme In the case where a defective subunit
cannot achieve a certain conformational state, this
may prevent the entire oligomer from interacting with
chaperones catalyzing later steps of folding In this
case, the wtASA subunit remains incompletely folded
and may be a substrate of UGGT
Heteromerization of defective polypeptides with
their normal counterparts has been demonstrated for
several membrane proteins that are defective in domi-nant genetic diseases For example, defective frizzled, a member of the Wnt signalling receptor family, forms oligomers in the ER and can retain wild-type frizzled
in the ER [21] Similar findings were reported for a kidney anion exchanger defective in renal tubular aci-dosis [22], for aquaporins in dominant diabetes insipi-dus [23] and for the GABAA receptor subunit [24] Only for the GABAAreceptor subunit were the conse-quences of heterooligomerization examined in detail Comparable to ASA, defective GABAA receptor sub-units also form oligomers with wild-type subsub-units, leading to the degradation of the latter by ERAD MLD is an autosomal recessive disease because deg-radation of wtASA induced by defective ASA has no biological consequence This is expected because indi-viduals with only 5–10% of the average ASA activity
of the normal population are healthy [25] Obviously, even low ASA activity maintains a normal catabolism According to the results obtained in the present study,
we would predict that, in carriers of defective ASA alleles, a fraction of wtASA will be degraded This fraction, however, does not suffice to lower the activity
to less than 10%, which would be necessary for the disease Accordingly, the present study did not aim to reveal mechanisms causing MLD Rather, the well characterized dimerization⁄ octamerization status of ASA, the availability of various structure-sensitive antibodies and defective enzymes, as well as the known 3D structure, all qualify this protein as an ideal tool for investigating the basic aspects of the oligomeriza-tion of proteins in vivo in more detail
Materials and methods Materials
Cell culture media and supplements were obtained from Invitrogen GmbH (Darmstadt, Germany) DNA restriction and modifying enzymes were purchased from Fermentas (Sankt Leon-Rot, Germany) [35S]methionine (specific activ-ity > 39 TBqÆmmol)1) was from Hartmann Analytik GmbH (Karlsruhe, Germany) Isolation of plasmids was performed using the QIA-Plasmid Midi Kit Qiagen GmbH (Hilden, Germany) in accordance with the manufacturer’s instructions The preparation and characterization of the moab A2 has been described previously [20] Hybridoma 12JA5 expressing HA antibody and 9E10 expressing Myc antibody, were cultured in RPMI medium containing 10% fetal bovine serum The HA antibody was purified from the medium by affinity chromatography using protein A sepha-rose; the Myc antibody was purified by protein G sepharose from GE Healthcare GmbH (Munich, Germany)
Trang 9Generation of HA and Myc tagged hASA
To generate HA-tagged hASA proteins, hASA cDNA was
amplified via PCR from a pBEH expression vector that
contains the hASA cDNA [26,27] using the primers:
forward: 5¢-dAAAGAATTCAAGCGTAATCTGGAACA
TCGTATGGGTAGGCATGGGGATCTGGGCAATG-3¢,
reverse: 5¢-dTTTGAATTCCATGTCCATGGGGGCACC
GCGGTC-3¢ The PCR product was cloned via EcoRI
restriction sites into the expression vector pBEH To
gener-ate wtASA-Myc proteins, oligonucleotides containing the
sequence of the Myc tag were generated: a BamHI
restric-tion site was integrated upstream of the Myc sequence and
a HindIII restriction site was integrated downstream of the
sequence Via these restriction sites, the Myc sequence was
cloned into the pBEH vector hASA cDNA was amplified
via PCR from the pBEH expression vector using the
prim-ers: forward: 5¢-dAAAGGATCCGGCATGGGGATCTGG
GCAATG-3¢, reverse: 5¢-dTTTGAATTCCATGTCCATGG
GGGCACCGCGGTC-3¢ The hASA was cloned via EcoRI
and BamHI sites into the Myc containing pBEH expression
vector
DNA transfection and ASA activity determination
Transfection of expression plasmids into BHK cells was
performed with ExGen 500 (Fermentas) Twenty four
hours prior to transfection, 8· 104
per 4· 105
BHK cells were seeded onto 24-well per six-well plates, respectively
For transfection, 22.5 lL per 121.5 lL 150 mm NaCl was
mixed with 0.5 lg per 2.7 lg of DNA, respectively; then
1.7 lL per 8.9 lL ExGen 500 was added and incubated
for 10 min after mixing Transfection solution was added
to 225 lL per 1215 lL DMEM containing 5% fetal
bovine serum and then added to the cells Fourteen hours
later, the DNA⁄ transfection reagent containing medium
was removed and replaced by serum-containing medium
To determine ASA activity, cells were harvested 48 h later
Twenty microliters of cell lysate, containing 20–50 lg of
protein, were incubated with 200 lL of substrate solution
(10 mm para-nitrocatecholsulfate in 0.5 m sodium acetate,
pH 5.0, 10% w⁄ v NaCl and 0.3% Triton X-100) for 30–
60 min at 37C The reaction was terminated by the
addi-tion of 500 lL of 1 m NaOH Absorpaddi-tion was measured
at 515 nm Protein content was determined with the DC
assay protein determination kit from Bio-Rad (Hercules,
CA, USA) in accordance with the manufacturer’s
instruc-tions
Metabolic labeling and immunoprecipitation
Protocols for metabolic labeling with [35S]methionine and
for subsequent immunoprecipitation of ASA have been
described in detail previously [28] Secretion of
newly-syn-thesized enzymes was enhanced by the addition of NH4Cl
in a final concentration of 10 mm The drug was also pres-ent during labeling periods Quantification of the precipi-tated proteins was performed after SDS⁄ PAGE with a Fuji bioimager (Fuji, Tokyo, Japan) Pixels of the corresponding polypeptide band were integrated by the software aida (raytest GmbH, Straubenhardt, Germany) After subtrac-tion of background values, the numbers obtained were taken as arbitrary values for the amount of 35S-labeled ASA
Immunoprecipitation and western blot Cell lysates of hASA expressing cells were incubated with either monoclonal hASA antibody A2 or polyclonal ASA antiserum or the monoclonal antibody against the Myc tag The antigen antibody complex was precipitated by Pansor-bin A in the case of hASA antibodies or protein G sepha-rose in the case of aMyc moab and washed three times with NaCl⁄ Pi After SDS⁄ PAGE, the proteins were blotted onto a nitrocellulose membrane hASA-HA was detected
by a biotinylated HA antibody and fluorophore-labeled streptavidin Detection was performed using a Li-Cor laser scanner (Li-Cor, Lincoln, NE, USA)
Biotinylation of HA antibody NaCl⁄ Pibuffered HA antibody (2 mgÆmL)1) was incubated with a 20-fold molar excess of EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) for 30 min The remaining free biotin was removed by fast desalting gelfil-tration on a SMART FPLC (GE Healthcare Europe, Frei-burg, Germany)
Acknowledgements This work was supported by a grant from the Euro-pean Leukodystrophy Foundation, the Deutsche Fors-chungsgemeinschaft and the BMBF The costs of publication of this article must therefore be marked as
an ‘advertisement’ in accordance with this fact
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