When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclona
Trang 1Degradation of arylsulfatase A in the endoplasmic reticulum
Peter Poeppel1, Matthias Habetha2, Ana Marca˜o3, Heinrich Bu¨ssow4, Linda Berna5
and Volkmar Gieselmann1
1 Institut fu¨r Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universita¨t Bonn, Germany
2 Zoologisches Institut, Christian-Albrechts-Universita¨t zu Kiel, Germany
3 Instituto de Biologia Molecular e Celular, University of Porto, Portugal
4 Institut fu¨r Anatomie, Rheinische-Friedrich-Wilhelms Universita¨t Bonn, Germany
5 Institute of Inherited Metabolic Disorders, Charles University, Prague, Czech Republic
Lysosomal storage diseases comprise a group of about
40 disorders, in most cases caused by the deficiency of
a lysosomal enzyme involved in the degradation of,
for example, lipids, glycosaminoglycans and
oligo-saccharides Much effort has been devoted to the
identification of disease causing mutations in these dis-orders Thus, a multitude of mutations has been identi-fied in recent years (for example [1]) Only a fraction
of missense mutations, however, has been analysed
at the biochemical level in order to understand the
Keywords
ERAD; proteasomal degradation;
arylsulfatase A; metachromatic
leukodystrophy
Correspondence
V Gieselmann, Institut fu¨r Physiologische
Chemie, Rheinische-Friedrich-Wilhelms
Universita¨t Bonn, Nussallee 11, 53115
Bonn, Germany
Fax: +49 22 873 2416
Tel: +49 22 873 2411
E-mail: gieselmann@institut.physiochem.
uni-bonn.de
Note
P Poeppel and M Habetha contributed
equally to this work.
(Received 29 October 2004, revised 14
December 2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04553.x
Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) Biosynthesis studies of ASA with vari-ous structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epi-topes form later, between 10 and 25 min When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes Eleven of the 12 mutants show partial expression of the early epi-topes, but only six of these show, in addition, incomplete expression of late epitopes In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation
of the late epitopes All mutant enzymes in which the amino acid substitu-tion prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER) In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER Thus, irrespective
of the missense mutation, the expression of epitopes forming late in biosyn-thesis correlates with the ability of the enzyme to leave the ER The degra-dation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER a1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes
Abbreviations
ASA, arylsulfatase A; ER, endoplasmic reticulum; Lc, lactacystin; Kif, kifunensine; SOV, sodium orthovanadate; PAO, phenylarsine oxide; OA, okadaic acid; Lp, leupeptin; DNM, deoxynojirimycin; a1-AT, a1-antitrypsin; MLD, metachromatic leukodystrophy; BHK, baby hamster kidney; DMEM, Dulbecco’s modified essential medium; FBS, foetal bovine serum; LDL-receptor, low density lipoprotein receptor.
Trang 2molecular basis of the enzyme deficiencies in greater
detail In many cases, missense mutations lead to an
arrest and more rapid degradation of the encoded
enzyme in the endoplasmic reticulum (ER) (for
exam-ple [2]) In this respect lysosomal storage diseases are
not special as this mechanism is responsible for protein
deficiencies in many diseases In fact, it has been
esti-mated that ER degradation is the most frequent cause
of protein deficiencies such that the term
‘conforma-tional diseases’ has been suggested [3]
The mechanisms of ER quality control, retention
and degradation have been investigated in recent years
(reviewed in [4]) Newly synthesized secretory,
mem-brane or lysosomal glycoproteins interact sequentially
with a number of membrane-bound or soluble
glyco-sidases and chaperones of the ER Modifications of
N-linked oligosaccharide side chains play a major role
in this process
The precursor of N-linked oligosaccharides is a Glc3
-Man9-GlcNAc2 dolichol pyrophosphate, from which
the sugars are transferred en bloc to Asn⁄ X ⁄ Ser(Thr) in
newly synthesized polypeptide chains within the ER
Trimming of the Glc3-Man9-GlcNAc2 side chains
begins shortly after synthesis by the ER
membrane-located glucosidase I to Glc2-Man9-GlcNAc2, followed
by trimming of an ER-localized soluble glucosidase II
to Glc1-Man9-GlcNac2 Glycoproteins bind to the
ER-resident lectins calnexin and calreticulin, via the
Glc1-Man9-GlcNAc2 oligosaccharide Glucosidase II
then removes the remaining terminal glucose with the
consequence that newly synthesized proteins no longer
bind to the lectins and leave the ER In case a protein
is not folded correctly, it is recognized by the
UDP-glu-cose:glycoprotein glucosyltransferase, which
reglucosy-lates the Man9-GlcNAc2 of misfolded proteins to
Glc1-Man9-GlcNAc2 [5] Consequently the protein can
bind to calnexin⁄ calreticulin again and remains in the
ER This loop can be repeated several times and may
enhance the chances of a protein folding correctly
Finally, a1,2-mannosidase I removes one mannose
[6,7] This removal of mannose by a1,2-mannosidase I
has been suggested to be a signal for proteasomal
de-gradation [7,8] The proteasome seems to be the major
pathway by which misfolded proteins are degraded,
although the existence of an as yet poorly characterized
nonproteasomal pathway has been demonstrated [6,7]
Metachromatic leukodystrophy (MLD) is a lysosomal
storage disorder which is caused by the deficiency of
arylsulfatase A (ASA) This enzyme catalyses the first
step in the degradation pathway of the
glycosphingo-lipid 3-O-sulfogalactosylceramide Deficiency of the
ASA causes lipid accumulation leading progressive
demyelination and various, ultimately lethal
neurologi-cal symptoms (reviewed in [1]) The gene of human ASA has been cloned and more than 80 mostly missense mutations were identified Some of these mutations were investigated more closely to reveal the effects of the amino acid substitutions on the mutant enzyme According to these results two main mechanisms cause ASA deficiency In about half of the examined cases the mutant enzymes are retained in the ER [2,9,10], in the other half, enzymes can leave the ER and be degraded after arrival in the lysosome [10–12] Whereas the latter mechanism has been investigated thoroughly in view
of potential therapeutic intralysosomal stabilization, nothing is known about the ER-associated degradation
as a cause of MLD Because it has been shown recently for Fabry disease [13]) another lysosomal storage disorder) the interference with the ER quality control mechanism can also be a therapeutic option, we decided
to examine more closely these mechanisms of enzyme deficiency in MLD
Results
Biosynthesis of wild-type ASA
To examine the early events in ASA biosynthesis in more detail, baby hamster kidney (BHK) cells were transiently transfected with a plasmid encoding human wild-type ASA cDNA Cells were pulse labelled with
Fig 1 Early stages of ASA biosynthesis BHK cells transiently expressing the human wild-type ASA cDNA were pulse labelled with 18.5 MBq [ 35 S]methionine for 2.5 or 5 min, respectively, and chased for the times indicated (0, 2.5, 5, 10 and 25 min) Cell homo-genates were split into eight aliquots and precipitated with preim-mune serum (I), a polyclonal ASA antiserum (II), or six different mAbs (A2, A5, B1, C, E and F), which are directed against five dif-ferent epitopes.
Trang 3[35S]methionine for 2.5 or 5 min and chased for up to
25 min (Fig 1) After harvesting, cell homogenates
were divided into eight aliquots, which were
immuno-precipitated with an ASA polyclonal antiserum or six
various mAbs [14] These mAbs recognize only native
ASA and are directed against different
structure-sensi-tive surface epitopes termed A, B, C, E and F [14]
After a pulse of 2.5 or 5 min, ASA can be readily
detected with the polyclonal antiserum As this serum
also recognizes denatured ASA, it precipitates ASA
irrespective of the enzyme’s three-dimensional
struc-ture After 2.5 and 5 min pulse only mAbs A2, A5 and
B1 recognize ASA, whereas no or minute amounts of
ASA are precipitated by the mAbs C, E and F
Epi-topes recognized by mAbs C, E and F start to develop
slowly within 10 min of chase and have matured after
another 15 min of chase Thus, in the early stages of
ASA biosynthesis, epitopes recognized by mAbs A2, A5
and B1 appear before those recognized by C, E and F,
demonstrating that ASA folds in a sequential manner
Recognition of amino acid-substituted ASAs
by mAbs
We have previously identified various missense
muta-tions in the ASA gene and we have examined the
biochemical effects of the corresponding amino acid
substitutions on ASA In a number of mutants, the
amino acid substitution causes an arrest of ASA in the
ER [2,9,10], whereas others can leave the ER [10–12]
We have expressed these mutant ASAs transiently in
BHK cells Cells were labelled for 3 h with [35
S]methi-onine and after harvesting, cell homogenates were again
divided into eight aliquots, which were
immunoprecipi-tated with the mAbs or polyclonal antiserum (Fig 2)
The analysis of 12 amino acid-substituted ASAs reveals
that, according to their reactivity with the mAbs, these
mutants can be divided into three groups One group
includes mutant ASAs which react weakly with mAbs
A2 and A5 and more strongly with B1 (Gly86Asp,
Tyr201Cys, Pro377Leu, Asp335Val, Pro136Leu,
Asp255His) None of these mutants, however, is
recog-nized by any of the antibodies C, E or F Substituted
ASAs of the second group (Gly309Ser, Glu312Asp,
Arg84Gln, Arg370Gln, Arg370Trp) react slightly better
with A2, A5 and B1 and react) although weakly )
with at least one of the mAbs C, E or F Finally, the
third group has only one member (Thr274Met) which
ASA is not recognized by any of the mAbs
In a previous publication we located the epitopes
recognized by the various mAbs (Table 3 in [14])
According to these data amino acid residues 85 and 86
may be part of the epitope recognized by mAbs A2
and A5, and amino acid residues 202–206 by mAb C, respectively For this reason the reduced reactivity of mAbs A2 and A5 with Gly86Asp and Arg84Gln sub-stituted ASA and mAb C with the Tyr201Cys substi-tuted ASA, respectively, may reflect changes in the epitopes rather than conformational alterations We could show in the meantime, however, that amino acids 202–206 are not part of the epitope recognized
by mAb C (P Poeppel, unpublished data), so that this cautionary notion does not apply to the immunopre-cipitation of Tyr201Cys substituted ASA with mAb C
Degradation of amino acid-substituted ASAs via the proteasome
In order to investigate the degradation pathway of amino acid-substituted ASAs in the ER, we used Ltk–
Fig 2 Immunoprecipitation of amino acid-substituted ASAs with structure-sensitive mAbs Wild-type ASA and 12 amino acid-substi-tuted ASAs were transiently expressed in BHK cells Cells were labelled for 3 h with 1.85 MBq [ 35 S]methionine, harvested and aliquots of cell homogenates were immunoprecipitated as des-cribed in Fig 1 + ⁄ – indicates whether or not the mutant enzymes according to previous publications (references in brackets; [2,9,10,25–28]) are retained in the ER Polypeptides of lower appar-ent molecular mass, which can be seen in some of the experi-ments are unrelated to ASA.
Trang 4cells which stably express the ER-retained ASA
mutant enzymes (Gly86Asp, Tyr201Cys, Pro377Leu,
Asp335Val, Pro136Leu, Asp255His, Thr274Met)
We selected those clones with a medium level of
over-expression and examined them by electron microscopy
for normal ER morphology, in order to exclude the
possibility that enzymes were being unphysiologically
overexpressed The examined cells showed an ER with
normal morphology (results not shown) Stably
trans-fected Ltk–cells were pulse labelled for 2 h and chased
for various time periods to determine the half-life of the
individual enzymes According to these experiments,
chase times were chosen for the following experiments
so that in most cases about 80–90% of the enzyme was
degraded within the chase periods Various inhibitors
were added during pulse and⁄ or chase periods Inhibits
lactacystin (Lc) irreversibly the 20 S proteasome,
leu-peptin (Lp) is an inhibitor of cysteine and serine
prote-ases, and okadaic acid (OA), phenylarsine oxide (PAO)
and sodium orthovanadate (SOV) are phosphatase
inhibitors The latter two were used as it has been
repor-ted that misfolded a1-antitrypsin (a1-AT) mutants or
immunoglobulin chains can be stabilized by these
com-pounds [7,8] Lp has been shown to stabilize some
mutant ASAs, which are degraded in the lysosome [12]
Under the conditions of the experiment Lp should
not inhibit the proteasome and was used as a
nonproteasomal control inhibitor Figure 3 shows an
experiment performed with seven different amino
acid-substituted ASAs The results demonstrate that
all of these mutant ASAs can be partially stabilized
by proteasome inhibition and that the extent of
stabilization varies between the substituted enzymes
Other inhibitors, in particular phosphatase inhibitors,
showed no effect
Effects of glycosidase inhibitors on the stability
of amino acid-substituted ASAs
In order to elucidate the role of trimming reactions
of the N-linked oligosaccharide side chains in ER
associated degradation of mutant ASAs, stably
trans-fected Ltk– cells were incubated with
deoxynojirimy-cin (DNM), an inhibitor of ER glucosidases I and II
and with kifunensine (Kif), an inhibitor of ER
a1,2-mannosidase I Cells were pulse labelled for 2 h and
chased for various times, depending on the half-life
of the mutants (Fig 4) The mutant ASAs were
sta-bilized by Kif, whereas inhibition of glucosidases I
and II causes a more rapid degradation Thus, all
substituted enzymes showed a uniform pattern of
stabilization or more rapid degradation upon addition
of inhibitors
Influence of ER a1,2-mannosidase I inhibition
on ER exit of amino acid-substituted ASAs
In order to investigate whether stabilization of mutant ASAs through inhibition of ER a1,2-mannosidase I via Kif can lead to an enhanced exit of mutant enzyme from the ER, stably transfected Ltk– cells were pulse-labelled for 15 h in the presence of Kif and⁄ or ammo-nium chloride After leaving the ER, lysosomal enzymes including ASA are specifically recognized by
a phosphotransferase in the Golgi apparatus [14] This enzyme initiates the phosphorylation of mannose in the N-linked oligosaccharide side chains of lysosomal enzymes, yielding mannose-6-phosphate (M6P) In the trans-Golgi these M6P residues bind to M6P receptors, which mediate the further vesicular transport of lyso-somal enzymes from the Golgi to the lysosomes Ammonium chloride interferes with this sorting and causes increased secretion of newly synthesized lyso-somal enzymes into the medium [15] Thus, if newly synthesized lysosomal enzymes appear in secretions in the presence of ammonium chloride, they must have left the ER The addition of ammonium chloride cau-ses secretion of wild-type ASA to the medium, whereas
it has no effect on ER-retained mutant enzymes Figure 5 shows Pro377Leu-substituted ASA as an example Also the stabilization of amino acid-substi-tuted ASA with Kif and simultaneous addition of ammonium chloride does not cause increased secretion into the medium, indicating that stabilization of enzymes does not lead to an escape from quality con-trol mechanisms and increased exit of the ER Only after prolonged exposure can minute amounts of mutant ASAs be detected in the medium, showing a marginal effect of Kif We estimate that this accounts for less than 5% of the enzyme synthesized during the pulse period
Discussion
Missense mutations are by far the most frequent type
of mutations in the ASA gene [1] The effects of these mutations have been shown to be rather uniform Either the amino acid substitutions lead to an arrest of the mutant enzyme in the ER, or the enzyme is degra-ded in the lysosome after correct sorting [2,9–12] Here
we have investigated wild-type and mutant ASAs by immunoprecipitation with six structure-sensitive mAbs These mAbs have recently been shown to recognize five different ASA epitopes, termed A to F These epi-topes, which were recently delimited more closely [14], depend on the native structure of ASA Examinations
of the early biosynthetic events reveal that epitope B
Trang 5forms rapidly after synthesis Already after a 2.5-min
pulse, the newly synthesized ASA is efficiently
precipi-tated by mAb B1 At the same time point precipitation
with mAbs A2 and A5 is possible but is less efficient,
indicating that the epitope may be less matured than
the B1 epitope
Figure 1 shows that the ratio of the signals obtained
with mAb A2⁄ A5 and B1 is constant up to 10 min of
chase (densitometric analysis, data not shown),
indica-ting that no further maturation of epitopes A2⁄ A5
occurs within this time period Epitopes C, E and F
are only weakly expressed until 10 min and mature
between 10 and 25 min after synthesis The maturation
of these late forming epitopes is accompanied by a further maturation of epitopes A2 and A5 After
25 min of chase, precipitation with mAbs A2 and A5
is almost as efficient as with mAb B1 The location of epitopes suggests that folding of ASA starts within a central part of the molecule [14] This is accompanied
by a partial expression of epitopes in the N-terminal part The C-terminal part folds late in biosynthesis, but its folding is not an isolated event, because epi-topes A2 and A5 mature concomitantly Studies on low density lipoprotein receptor (LDL-receptor) folding
Fig 3 Effects of protease or phosphatase
inhibitors on the stability of mutant ASAs.
Ltk – cells stably expressing the indicated
amino acid-substituted ASAs were
incuba-ted in the presence of various inhibitors
(Lc, Lp, OA, PAO, SOV) Cells were pulse
labelled for 2 h and chased for various times
depending on the half-life of the respective
mutant (Asp335Val, 4.5 h; Gly86Asp,
05.25 hours; Pro377Leu, 4 h; Tyr201Cys,
6 h; Thr274Met, 4.5 h; Asp255His, 4.5 h;
Pro136Leu, 8 h) After the chase ASA was
immunoprecipitated from the homogenates
with the polyclonal ASA antiserum
Precipi-tated ASA was quantified after SDS ⁄ PAGE
with a bio-imaging analyser (Fujifilm)
Col-umns show mean and SD of arbitrary units
of quadruple experiments Under each
dia-gram representative immunoprecipitates are
shown.
Trang 6have shown recently [16] that its folding does not
proceed in a vectorial, domainwise process from the N
terminus to the C terminus Instead, folding occurs via
intermediates with disulfide bridges involving distant
parts of the protein In addition, the N-terminal part
of the LDL-receptor forms late in biosynthesis Data
on ASA are in agreement with this folding scheme
Early detectable epitopes are constituted by amino acid
residues between positions 165 and 240 in the central
part of the protein [14] N-terminal epitopes are also
detectable at an early stage, but do not mature before
the C-terminal part of the protein folds correctly As
in case of the LDL-receptor, this suggests interactions
of distant parts of ASA during folding
In addition to wild-type ASA, we also
immunopre-cipitated 12 mutant ASAs, whose underlying missense
mutations were previously found in MLD patients
Since the mAbs only recognize the native wild-type
enzyme, we reasoned that the reactivity with the mAbs
should provide a measure of the structural integrity of
the substituted enzymes Surprisingly, the mutant
enzymes did not show an individual reaction pattern
but according to their immunoprecipitation pattern
they can be classified into three groups One group has
only one member, mutant Thr274Met, which does
not react with any of the mAbs but is readily precipi-table with the polyclonal antiserum This reveals
a severe misfolding of this mutant The second group of mutants (Gly86Asp, Tyr201Cys, Pro377Leu, Asp335Val, Pro136Leu, Asp255His) reacts partially with antibodies recognizing the early epitopes A and B and not with those recognizing the late epitopes C, E and F Interestingly, mutant ASAs of these two groups are completely retained in the ER Retention in the
ER due to incorrect folding leads to repetitive regluco-sylation and binding to the calnexin and calreticulin chaperones Finally, a mannose is removed, which is considered to be a signal for reverse transport out of the ER into the cytosol After the transfer of the mis-folded protein into the cytosol, N-glycans are removed and the protein is degraded by the proteasome
The last group is comprised of mutant ASAs which form, at least partially, one or more of the late epitopes
C, E and F Thus, except for Thr274Met, all mutant enzymes express partially the early epitopes whereas only a fraction expresses the late ones This suggests that in general the latter are more sensitive to amino acid substitutions, irrespective of their localization Also none of the mutants expresses the late epitopes only, or to a larger extent, than the early epitopes This
Fig 4 Effects of glycosidase inhibitors on stability of mutant ASAs Ltk–cells stably expressing the indicated amino acid-substi-tuted ASAs were incubated in the presence
of the two inhibitors DNM or Kif Cells were pulse labelled for 2 h and chased for various times depending on the expression and the half-life of the respective mutants
(Asp335V-al, 4.5 h; Gly86Asp, 5.25 hours; Tyr201Cys,
5 h; Thr274Met, 4.5 h; Asp255His, 4.5 h; Pro136Leu, 4.5 h) After the chase, ASA was immunoprecipitated from the homogen-ates with the polyclonal ASA antiserum Precipitated ASA was quantified after SDS ⁄ PAGE with a bio-imaging analyser (Fujifilm) Columns show mean, minimal and maximal deviation of arbitrary units of two independent experiments Under each dia-gram representative immunoprecipitates are shown.
Trang 7indicates that the formation of the epitopes of ASA is
sequential in two aspects: (a) two epitopes (A, B)
form rapidly after translation, whereas others need
several minutes to mature; and (b) formation of early
epitopes is a prerequisite for the maturation of the late
epitopes
Interestingly, none of the mutants that react with at
least one of the antibodies C, E or F is retained in the
ER (Gly309Ser, Glu312Asp, Arg370Gln, Arg84Gln,
Arg370Trp) Our results suggest that enzymes have
reached a folding state which suffices to pass the ER
quality control, when they express at least epitope C
partially (Arg370Gln and Arg370Trp) In a separate
study we have identified two additional mutations
(Phe219Val, Pro425Thr) that generally also fit into this
pattern ([11] A Marca˜o, unpublished data) It should
be mentioned that one of these mutations (Phe219Val)
was found in a patient with an unusual phenotype and
encodes an enzyme that, like Thr274Met, does not
react with any of the mAbs This mutant ASA,
how-ever, can leave the ER to an extent of about 20% of
the newly synthesized enzyme, the remainder is
retained in the ER
Recently it was reported that a certain mutant of a1-AT is retained in the ER and degraded by nonpro-teasomal pathways [7] This mutant could be stabilized
by the addition of phosphatase inhibitors PAO and SOV The existence of such a pathway is supported by the fact that phosphatase inhibitors can also inhibit immunoglobulin chain degradation in the ER [8] Here
we examined whether different ASA mutants, which are retained in the ER, show differences in the ER degradation For that purpose we have investigated the influence of various protease and glycosidase inhib-itors on the stability of the substituted enzymes All these mutants are partially stabilized by the protea-somal inhibitor Lc but not by the serine and cysteine protease inhibitor Lp, or any of the phosphatase inhib-itors All mutant ASAs seem to be uniformly degraded via the proteasome; there is no indication that different mutants may use different degradation pathways It should also be mentioned, however, that in none of the cases could we achieve a full stabilization upon proteasome inhibition In fact the degree of stabiliza-tion in some mutants (e.g., Thr274Met, Pro136Leu) was rather weak Although the lack of full-scale stabil-ization was unchanged when we increased the protea-some inhibitor concentration (data not shown), we cannot exclude that the proteasome was inhibited only partially Nevertheless, the lack of stabilization by the phosphatase inhibitors indicates that recently detected nonproteasomal pathways [7,8] do not contribute to ASA degradation in the cell type used in this examina-tions
Proteins may be degraded in an ubiquitin-independ-ent way by the 20S proteasome In various experimubiquitin-independ-ents (not shown) we failed to detect ubiquitinylation of the ASA mutants, suggesting that they may be degraded
in a ubiquitin-independent way by the 20S proteasome [17]
Glucosidases I and II, as well as ER a1,2-mannosid-ases, play a role in the targeting of misfolded proteins
in the ER [6–8,18–20] For this reason we investigated the influence of glucosidase and mannosidase inhibi-tion on the mutant ASAs (Gly86Asp, Tyr201Cys, Asp335Val, Pro136Leu, Asp255His, Thr274Met) In these experiments all of the mutant ASAs behaved rather uniformly They could all be stabilized by Kif,
an ER a1,2-mannosidase I inhibitor In all cases the degradation was enhanced when glucosidases I and II were inhibited by DNM Increased degradation upon inhibition of glucosidases and stabilization by inhibi-tion of mannosidases is a common phenomenon and has been demonstrated for various misfolded proteins [6–8,21,22] The behaviour of ASA mutants in the
ER in the presence of various inhibitors is identical,
Fig 5 Effects of Kif on secretion of mutant ASAs Ltk – cells stably
expressing wild-type ASA and Pro377Leu substituted ASA were
incubated in the presence of Kif, ammonium chloride (NH 4 Cl) or a
combination of both compounds Cells were pulse labelled for
16 h After the labelling ASA was immunoprecipitated from the
homogenates and secretions with the polyclonal ASA antiserum.
The right panel shows an overexposed sample of the
immunopre-cipitates from the secretion of Pro377Leu, which demonstrates
that only low amounts of Kif stabilized ASA appear in the medium.
The same experiment was performed with all ER retained ASAs, all
showed identical results.
Trang 8showing that all mutants interact uniformly with
com-ponents of the ER degradation pathway independent
of the underlying mutations
The interference with ER quality control may open
new therapeutic strategies in the treatment of genetic
diseases Thus, it has been shown that secretion of an
otherwise ER retained mutant protein, an a1-AT, is
enhanced upon inhibition of ER a1,2-mannosidase I
[23] For that reason we have examined whether in
principal any of the mutant ASAs can be delivered
from the ER upon inhibition of the degradation
path-way through inhibition of ER a1,2-mannosidase I
Cells were treated with Kif and⁄ or ammonium
chlor-ide The latter interferes with lysosomal enzyme sorting
in the Golgi, so that newly synthesized lysosomal
enzymes appear in the medium In the case of mutants,
the appearance in the medium is thus an indicator that
the enzyme has left the ER In none of the analysed
mutants, however, does treatment with Kif lead to a
substantial increase of ASA in the medium Thus, in
case of ASA, inhibition of the degradation pathway
does not lead to enhanced secretion, which suggests it
will not be a therapeutic option for MLD
Experimental procedures
Materials, enzymes, chemicals, antibodies
Enzymes used for DNA modification or synthesis were
from New England Biolabs (Frankfurt am Main, Germany)
or Invitrogen (Karlsruhe, Germany) [35S]Methionine
(spe-cific activity > 39 TBqÆmmol)1) was from Amersham
Bio-sciences (Buckinghamshire, UK) Oligonucleotides were
from MWG Biotech (Ebersberg, Germany) or Eurogentec
(Seraing, Belgium) The preparation and characterization of
the mAbs has been described previously [14]
Cell culture and transfection
Mouse fibroblast Ltk– cells (Ltk–) and BHK cells were
(DMEM) supplemented with 5 or 10% fetal bovine serum
(FBS), penicillin and streptomycin For transient
transfec-tions, BHK cells were transfected by LipofectamineTM
(Gibco, Karlsruhe, Germany) Cells (2· 105
) were plated onto a 3.5-cm cell-culture dish Next day, medium was
removed and cells were washed with DMEM devoid of
supplements Plasmid DNA (2 lg) was mixed with 750 lL
DMEM containing 5 lL LipofectamineTMreagent After a
30-min incubation, the DNA–LipofectamineTM complexes
were added to the cells in a total volume of 1.5 mL After a
5-h incubation, the LipofectamineTM-containing medium
harvested and analysed for enzyme activity and protein concentration 48 h after transfection In case of stable transfections, 1.2· 106
Ltk–cells were plated onto a 6-cm cell-culture dish The next day, medium was removed and 1.5 mL DMEM containing 5% FCS, penicillin and strepto-mycin was added Plasmid DNA (5 lg) was mixed with
300 lL 150 mm NaCl After vortexing, 15.5 lL ExGen 500 reagent (Fermentas, St Leon-Rot, Germany) was added and incubated for 10 min This solution was added to the cells and left for 7 h, after which the ExGen 500-containing medium was removed and replaced by DMEM in the pres-ence of 5% FBS, penicillin and streptomycin In the case of stable transfections one tenth of the transfected plasmids was pSV2neo carrying a neomycin-resistance gene Cells were selected in 800 lgÆmL)1 G-418 (Invitrogen) and single colonies were screened for expression of ASA mRNA by northern blot and protein by western blot analysis ASA activity was measured with the artificial substrate 10 mm p-nitrocatecholsulfate in 170 mm NaCl, 500 mm sodium acetate pH 5, 0.3% Triton X-100 and 1 mgÆmL)1 BSA
200 lL of substrate solution was incubated with 5–50 lg protein of cell homogenates Reaction was performed at
37C for various time periods and stopped with 500 lL of
1 m NaOH Absorption was read at 515 nm To obtain measurements in the linear range, only samples with an extinction below 0.7 were included; otherwise the determin-ation was repeated with shorter incubdetermin-ation times
Metabolic labelling and immunoprecipitation
Metabolic labelling and immunoprecipitation have been described in detail elsewhere [24] In the experiments in which the degradation pathway of the mutant enzymes were investigated the following inhibitors and final concen-trations were used: lactacystin (Lc) 25 lm (Calbiochem, Bad Soden, Germany), kifunensine (Kif) 100 lm (Calbio-chem), sodium orthovanadate (SOV) 50 lm (Sigma), phenyl-arsine oxide (PAO) 800 nm (Sigma, Munich, Germany), okadaic acid (OA) 100 nm (Calbiochem), leupeptin (Lp)
(kindly provided by E Bause, Institut fu¨r Physiologische Chemie, Rheinische-Friedrich-Wilhelm Universita¨t Bonn, Germany) Lc was present during the pulse and chase peri-ods, the others only during the chase periods In the experiments in which the secretion of newly synthesized enzymes was enhanced by the addition of NH4Cl, the drug was added to a final concentration of 10 mm and was pre-sent during labelling periods When immunoprecipitation was performed under nondenaturing conditions with the mAbs, SDS was omitted from all solutions and the immu-noprecipitation procedure was modified accordingly In this case, cells were harvested in 50 mm Tris⁄ HCl pH 7.0, 0.2% Triton X-100 containing 25 lgÆmL)1 leupeptin, 1 mm phenylmethanesulfonyl fluoride, 5 mm iodoacetamide and
Trang 95 mm EDTA After removing debris by centrifugation at
10 000 g for 10 min the supernatants were adjusted to 5%
BSA, 0.2% Triton X-100, 0.1% sodium deoxycholate
and 150 mm NaCl (buffer A) The adjusted supernatants
were preabsorbed twice for 30 min with 100 lL of a 10%
Staphylococcus aureus(Calbiochem) suspension, which was
removed by centrifugation at 10 000 g for 10 min mAbs
and antisera were added to the cleared supernatants and
incubation continued for 16 h at 4C Five micrograms of
an anti-mouse IgG, raised in rabbits, was added to the
samples containing the mAbs and incubation proceeded for
another 2 h ASA–antibody complexes were collected with
25 lL of a 10% S aureus suspension for 30 min S aureus
pellets were washed twice in ice-cold buffer A and once
with NaCl⁄ Pi The quantification of precipitated proteins
was performed after SDS⁄ PAGE, with a bio-imaging
ana-lyser (Fujifilm, Dusseldorf, Germany)
Acknowledgements
This work was supported by a grant of the Deutsche
Forschungsgemeinschaft We thank Dr E Bause for
providing deoxynojirimycin
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