Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 276-282
Janua~ 15,1992
H U M A N C A T H E P S I N D P R E C U R S O R IS A S S O C I A T E D W I T H A 60 k D a G L Y C O S Y L A T E D P O L Y P E P T I D E
Susanne
Gr~ssel
and Andrej
Hasilik
Institut f~r P h y s i o l o g i s c h e Chemie und P a t h o b i o c h e m i e W a l d e y e r s t r a s s e 15, D - W 4400 Mdnster, FRG
Received November 15, 1991
SUMMARY Human cathepsin D is s y n t h e s i z e d as a 53 kDa precursor. Most of it is s e g r e g a t e d into lysosomal c o m p a r t m e n t s and subjected to a p r o t e o l y t i c fragmentation. Using a c r o s s - l i n k i n g reagent we s h o w t h a t a large p r o p o r t i o n of the p r e c u r s o r is a s s o c i a t e d with a distinct protein which - under denaturing and reducing c o n d i t i o n s - is c h a r a c t e r i z e d as a 60 kDa g l y c o p e p t i d e . S t u d i e s on c e l l s c u l t u r e d in the p r e s e n c e of d r u g s k n o w n to a f f e c t the i n t r a c e l l u l a r t r a n s p o r t (deoxynojirimycin, b r e f e l d i n A and NH4CI) indicated that the a s s o c i a t i o n with cathepsin D p r e c u r s o r occurs e a r l y a f t e r the s y n t h e s i s and is at least p a r t i a l l y m a i n t a i n e d after secretion. @1992AcademicPress,Inc.
Cathepsin
D and s e v e r a l
synthesized
as p r e c u r s o r s ;
endoplasmic
reticulum
which may Removal
influence
of g l u c o s e
other soluble reviewed
nascent its
residues
in r e f e r e n c e s
cathepsin
folding
lysosomal
D becomes
and the
the p r e c u r s o r from the endoplasmic r e t i c u l u m in a c i s - G o l g i mannose
compartment
6-phosphate
is n e e d e d
receptors
for
(1,2).
ments.
of l y s o s o m a l
enzyme
for the e x p o r t
interaction network.
to
lysosomal
with These in the
compart-
Because of the m o d i f i c a t i o n and s e g r e g a t i o n reactions,
precursors
participate
in a n u m b e r
of t r a n s i e n t
T h e s e m i g h t be r e v e a l e d by c o v a l e n t l y sors to the interacting proteins. reagents
cathepsin
0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
276
the
interactions.
cross-linking
the p r e c u r -
We show that using b i f u n c t i o n a l
D p r e c u r s o r can be captured
a 60 kDa glycopeptide.
of
(3). P h o s p h o r y l a t i o n its
in the t r a n s - G o l g i
precursors
In the
transport.
receptors and alternative targeting m e c h a n i s m s p a r t i c i p a t e delivery
are
N-glycosylated
subsequent
is a p r e r e q u i s i t e
enzymes
in a complex with
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS Cells, culturing and metabolic labeling. Human promonocytes U937 w e r e m a i n t a i n e d at 37°C in a m i x t u r e of air and CO 2 (19:1) in RPMI 1640 m e d i u m (Gibco BRL, Eggenstein, FRG) s u p p l e m e n t e d with i00 u n i t s / m 1 p e n i c i l l i n , i00 ~ g / m l s t r e p t o m y c i n and 10%(v/v) foetal bovine serum (Boehringer-Mannheim, Mannheim, FRG) that had been heat-inactivated. Prior to labeling the cells were incubated with 100 nM calcitriol (l,25-dihydroxycholecalciferol, donated by Dr. M.R. Uskokovi6, Hoffman-La Roche, Nutley, NJ) for 3 to 4 days (4). M e t a b o l i c l a b e l i n g was p e r f o r m e d w i t h 1.8 M B q of e i t h e r [ 3 5 S ] m e t h i o n i n e (Amersham Buchler, B r a u n s c h w e i g , FRG) or a 35Slabeled amino acid mixture (Tran 35 S-label from ICN Biomedicals, Irvine, CA) in 1 ml m e d i u m c o n t a i n i n g 0.5 or 1 x 106 cells in a modified RPMI 1640 medium. This medium was prepared without methionine and cysteine and with 4% (v/v) dialyzed heat inactivated foetal b o v i n e serum. B r e f e l d i n A, a gift from Dr. A. T a k a t s u k i (The Institute of Physical and Chemical Research, Saitama, Japan) was added at a c o n c e n t r a t i o n of 2.5 ~g/ml at the b e g i n n i n g and after 4 h of labeling that was continued for another 4 h. Deoxyn o j i r i m y c i n , a gift from Drs. G. S c a n g o s and D. S c h m i d t (Bayer AG, W u p p e r t a l , FRG) was a d d e d 1 h p r i o r to the l a b e l i n g at a c o n c e n t r a t i o n of 20 mM. Cross-linking and immunoprecipitation of cathepsin D. Prior to the c r o s s - l i n k i n g the cells w e r e centrifuged, w a s h e d and resusp e n d e d in 1 ml 0.i M 3 - ( N - m o r p h o l i n o ) p r o p a n e s u l p h o n i c acid-KOH buffer, pH 7.4. The reagents, dithiobis(succinimidylpropionate), disuccinimidyl suberate and ethylene g l y c o l b i s ( s u c c i n i m i d y l s u c c i nate) were added from 50 mM stock solutions in dimethylsulfoxide to a final concentration of 1 mM to either the cell suspension or the medium. These reagents were purchased from Pierce, Rockford, IL. The i n c u b a t i o n w a s p e r f o r m e d for i0 m i n at 37°C and was t e r m i n a t e d by adding 0.25 ml freshly p r e p a r e d m i x t u r e of 0.7 M NaCl, 0.5% s o d i u m d o d e c y l sulfate, 5% T r i t o n X-100, 2.5% Nadeoxycholate, 25 mg/ml b o v i n e serum albumin, 5 mM MgCI2, 25 mM iodoacetamide, 5 mM p h e n y l m e t h y l s u l f o n y l fluoride, 0.i m g / m l DNase I and 50 mM Na-phosphate, pH 7.4. The mixture was incubated 15 min at room temperature, freeze-thawed three times and centrifuged 1 h at 40,000 xg. The supernatant was supplemented with 1.5 mM ethylenediamine tetraacetate and cathepsin D was p r e c i p i t a t e d in the presence of affinity purified rabbit anti-human cathepsin D antibody as p r e v i o u s l y described (5). Treatment with glycosidases and separation of the labeled polypeptides. The i m m u n o p r e c i p i t a t e s w e r e w a s h e d w i t h several solutions (5) and s o l u b i l i z e d by h e a t i n g in the p r e s e n c e of detergents. The composition of mixtures used for the incubations with glycosidases was described previously (6). E n d o - ~ - N - a c e t y l g l u c o saminidase H and glycopeptidase F were from Boehringer-Mannheim. Prior to electrophoresis in polyacrylamide gels (5,7) the samples were heated at 95°C in the presence of 1% sodium dodecyl sulfate with or without 20 mM dithiothreitol as indicated. The gels were processed as previously (5,7).
RESULTS U937 the
cells
expression
labeled obtain
were of
cultured cathepsin
by c u l t u r i n g concommittant
for
with D.
several
radioactive 277
i00 The
nM c a l c i t r i o l cells
hours
with
labeling
were
to
enhance
metabolically
[35S]methionine of
the
to
precursor,
Vol. 182, No. 1, 1992
intermediate the
cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and large mature
were
treated
with
led proteins
were
extracted
polypeptides
were
then
in p o l y a c r y l a m i d e detected
by
cinimidyl
gel
subunit
of c a t h e p s i n
cross-linking with
isolated
fluorography.
When
or
when
in t h e p r e s e n c e
the cells were they
were
treated
treated
bis(succinimidylpropionate)
and the electrophoresis
in n o n - r e d u c i n g
a major
sor was was
conditions,
observed
used
and
(Fig.
the
i, l a n e s
relative
3 and
immunoprecipitates
C0 DSS DSP EGS DTT + + + +
C0 DSP - -
were
Cells + - +
of
the
1
2
-
labe-
separated o f SDS, with
disuc-
the
precur-
latter
reagent
cathepsin
Medium + + - + - + -
9 6
46 30
Q
1
2
3
4
5. 6
~2)
3
4
5
6
7
8
Fiq. I. Composition of the i~munoprecipitates of cathepsin D from c a l c i t r i o l - t r e a t e d m e t a b o l i c a l l y labeled U937 cells. Prior to e x t r a c t i o n the cells were incubated for 7 m i n at 37°C w i t h o u t (Co) or with 1 mMdithiobis(succinimidylpropionate) DSP, disuccinimidyl suberate, DSS, or ethylene glycolbis(succinimidysuccinate) EGS. Cathepsin D was immunoprecipitated and the precipitates were denatured at 95°C in the presence of sodium dodecyl sulfate with or without i0 mM. The samples were either reduced or not with i0 mM d i t h i o t h r e i t o l (DTT) and s e p a r a t e d by p o l y a c r y l a m i d e gel electrophoresis. The gels were processed for fluorography. The arrow indicates the material that is released from a cross-linked aggregate together with the cathepsin D precursor (P). The positions of the intermediate (I) and large mature (LM) polypeptides of cathepsin D are indicated. The standards were phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase. Fiq. 2. Detection of a cathepsin D precursor-binding protein by c r o s s - l i n k i n g w i t h dithiobis(succinimidylpropionate) in cells cultured in the presence of brefeldin A (BFA) and in the medium of cells cultured in the presence of i0 mM NH4CI. The immunoprecipitates were reduced. The arrows indicate the position of the 60 kDa protein that is associated with cathepsin D precursor in cells and the medium. P, I and LM refer to the precursor, intermediate and large mature polypeptides of cathepsin D. 278
and
dithio-
(~
97.4 69
D
was performed
reduced,
+ +
the
of
Cathepsin
with
loss
6). W h e n
BFA NH~tCI
(kDa
and
mixture.
immunoprecipitation,
electrophoresis
suberate,
reagents
a detergent by
D. A l i q u o t s
D
Vol. 182, No. 1, 1992
polypeptides
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were detected
in a n o r m a l
proportion
to each o t h e r
and were a c c o m p a n i e d with a p o l y p e p t i d e w i t h an a p p a r e n t molecular mass of 58-64 kDa Apparently, aggregates
(Fig. i, lane 2).
at least a p o r t i o n of c a t h e p s i n D p r e c u r s o r forms
with
other
proteins.
Large
aggregates
are
obtained
after c r o s s - l i n k i n g with d i t h i o b i s ( s u c c i n i m i d y l p r o p i o n a t e ) .
These
aggregates
after
contain
denaturation
and
a distinct
reduction.
protein
It
which
is r e l e a s e d
is c h a r a c t e r i z e d
by
an a p p a r e n t
m o l e c u l a r mass of 58-64 kDa and will be referred to as the 60 kDa polypeptide.
In a p u l s e - c h a s e experiment a t r a n s i e n t r a d i o a c t i v e
labeling of this p o l y p e p t i d e was observed similar to that of the p r e c u r s o r c a t h e p s i n D (not shown). B r e f e l d i n A i n t e r f e r e s w i t h the v e s i c u l a r the e n d o p l a s m i c allows tion
r e t i c u l u m and the Golgi
for a partial
of
phosphorylation,
"uncovered"
cathepsin
D and
cross-linking
mannose
but
(8).
it p r e v e n t s
6-phosphate
its s u b s e q u e n t p r o c e s s i n g
with
transport
apparatus
between
This drug the
forma-
residues
in p r e c u r s o r
as w e l l
(9). W h e n the
dithiobis(succinimidylpropionate)
was
perfor-
med w i t h cells that had been labeled in the p r e s e n c e of b r e f e l d i n A, the
intensity
D and the
of the labeling of both the p r e c u r s o r
60 kDa p o l y p e p t i d e
2). W h e n
the
which
known
is
precursors
labeling to
was
was
performed
enhance
the
(7), the s e c r e t e d
of s o m e w h a t
(63-67
lane
Fig.2,
secretion
2,
of
cathepsin
lanes
1 and
of N H 4 C I ,
lysosomal
enzyme
D precursor was accompa-
larger
7) t h a n
(Fig.
in the p r e s e n c e
cathepsin
n i e d by a m a t e r i a l kDa;
enhanced
apparent
observed
molecular
mass
for the m a t e r i a l
that
could be c r o s s - l i n k e d to the intracellular precursor. After a treatment to i n t r a c e l l u l a r peptidase
of the p o l y p e p t i d e s
and s e c r e t e d
cathepsin
that were
cross-linked
D precursor with glyco-
F, their apparent m o l e c u l a r mass was reduced to appro-
ximately
55
cathepsin
D precursor
kDa.
The
polypeptides
that
were
cross-linked
in either cells or m e d i u m were
tive to e n d o - ~ - N - a c e t y l g l u c o s a m i n i d a s e
to
less sensi-
H than to g l y c o p e p t i d a s e F
(Fig. 3). It is likely that the g l y c o p e p t i d e s that are a s s o c i a t e d with
precursor
cathepsin
same polypeptides,
D in c e l l s
and
in t h e m e d i u m
are
but have different c a r b o h y d r a t e moieties.
c o u l d be e x p e c t e d for g l y c o p r o t e i n s
the This
in w h i c h c o m p l e x o l i g o s a c c h a -
rides are formed in the Golgi apparatus. Deoxynojirimycin the
transport
reticulum
of
interferes with the d e g l u c o s y l a t i o n and with
precursor
cathepsin
D from
the
endoplasmic
(3). If the labeling of U937 cells was p e r f o r m e d
279
in the
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cells DSP
-
Cells
+ +
-
-GPF+ -
Medium (NH4CI)
Cells
+ +
+ + + +
+ +
+
-
-
+ . . . . .
F~do H + -
1 2
3
4
5 6
-
+
-
+ - - -N-+
7
8
9 10 I1 12
1314
Fiq. 3. Cleavage and processing of the N-linked oligosaccharides in the 60 kDa g l y c o p e p t i d e in c a l c i t r i o l - t r e a t e d U937 cells. Cathepsin D was immunoprecipitated from cells that were labeled in the presence of brefeldin A alone (lanes 1-8 and 13) and of brefeldin A and deoxynojirimycin (DNM lane 14) or from the medium of cells that were labeled in the presence of i0 mM NH4CI (lanes 9-12). T h e c e l l s a n d t h e m e d i u m w e r e t r e a t e d w i t h d i t h i o bis(succinimidylpropionate) DSP as indicated. Aliquots of reduced immunoprecipitates were incubated without or with endo-~-N-acetylglucosaminidase H (Endo H) or glycopeptidase F (GPF) as indicated. The position of the 60 kDa glycopeptide in the non-digested samples is indicated by arrows.
presence linking
of
both
resulted
of a slightly din
A
drugs ting
alone the
the
that
complexes
and
larger glycopeptide (Fig.
the presence
A
deoxynojirimycin
in a c o p r e c i p i t a t i o n
precursor
that
teins
brefeldin
3,
lanes
13
cathepsin
processing
and
of d e o x y n o j i r i m y c i n .
accumulated
than
in t h e
precursor
In
also
the
glycoproteins Thus,
presence
the
both
indica-
inhibited
less mature
of this
of
larger
was
drug
D
of b r e f e l -
presence
slightly
cross-
cathepsin
in t h e p r e s e n c e
14).
D was
of both
with
the
in
glycopro-
also
formed
and could be cross-linked.
DISCUSSION
In r a b b i t m a c r o p h a g e s human
hepatoma
precursor These
results
cathepsin another could
form
be
D
(i0), m o u s e
cells HepG2 of c a t h e p s i n
(12)
association
D with
give us no information precursor
serves
fibroblastic
its
function.
We
expected
that
detected
by
cross-linking
280
have
as to w h e t h e r modification, putative and
(ii)
and
of p r e f e r e n t i a l l y
membranes
to
cells
been
the
reported.
the binding
of
targeting,
or
binding
components
coprecipitation
with
Vol. 182, No. 1, 1992
cathepsin
D
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in c e l l s
synthesizing
These conditions
are m e t
the
calcitriol.
presence
of
If t h e s e
dithiobis(succinimidylpropionate) i s o l a t e d by
cathepsin
D at a h i g h
in h u m a n p r o m o n o c y t e s
immunoprecipitation
U937
cells
cathepsin
are
rate.
cultured
treated
D precursor
in
with
can
be
in form of l a r g e a g g r e g a t e s .
If
the a g g r e g a t e s are treated with a reducing agent,
a 60 kDa glyco-
p e p t i d e is released along with p r e c u r s o r c a t h e p s i n D. This glycopeptide
c a n be e a s i l y
labeled
in the p r e s e n c e
detected
intracellular protein of c a t h e p s i n tected
transport
D precursor
(9).
in o t h e r h u m a n cells
carcinoma
MCF7,
MG-63
colon
least
a portion
of
the
60
in the m e d i u m
molecular
interme-
kDa
of
m a y be
glycopeptide
is
c a t h e p s i n D even a f t e r the s e c r e t i o n .
together with cathepsin
associated
to the
60 kDa g l y c o p e p t i d e
immunoprecipitation
apparent
cells
D. D u r i n g the m a t u r a t i o n
or the
After cross-linking
cells.
mammary
carcinoma
is not c r o s s - l i n k e d
site
associated with precursor
glycopeptide
can a l s o be de-
skin f i b r o b l a s t s , and
forms of c a t h e p s i n
D its b i n d i n g At
an a c c u m u l a t i o n
(not shown).
d i a t e and m a t u r e
destroyed.
(8) and i n d u c e s
including
The 60 kDa g l y c o p e p t i d e
cathepsin
are m e t a b o l i c a l l y interferes w i t h the
The g l y c o p e p t i d e
osteosarcoma
Caco-2 and HT-29
if t h e c e l l s
of b r e f e l d i n A which
with
mass
from N H 4 C l - t r e a t e d
cathepsin
than
after
U937
cells,
D and reduction,
D has
a slightly
cross-linking
the
larger
within
the
This and the r e d u c t i o n of its mass after a t r e a t m e n t with
g l y c o p e p t i d a s e F indicates that w i t h i n the Golgi apparatus the 60 kDa
glycopeptide
glycopeptide labeled
acquires
is isolated
F,
its
i n d i c a t e the p r e s e n c e
oligosaccharides.
When
the
from cells that have been m e t a b o l i c a l l y
in the p r e s e n c e
glycopeptidase
complex
of b r e f e l d i n A, apparent
and t h e n
molecular
mass
is
reduced
to
three N-linked
oligosac-
charides.
T r e a t m e n t with e n d o - ~ - N - a c e t y l g l u c o s a m i n i d a s e
H removes
probably
only
one
of
of a p p r o x i m a t e l y
incubated with
these
oligosaccharides
and
indicates
partial p r o c e s s i n g that may be expected after a p r o l o n g e d tion in the p r e s e n c e
of b r e f e l d i n A
(8).
In U937
a
incuba-
cells c a t h e p s i n
D p r e c u r s o r has been shown to become p a r t i a l l y p h o s p h o r y l a t e d but its p h o s p h a t e
residues
have
not been
"uncovered"
i n c u b a t i o n in the p r e s e n c e of b r e f e l d i n A
during
a 4 h
(9).
We have not e x a m i n e d if the c r o s s - l i n k e d a g g r e g a t e s are bound to a membrane.
Perhaps
c o n t r i b u t e to that ment w i t h
large aggregates
of c a t h e p s i n
D precursor
fraction of the p r e c u r s o r that after a treat-
saponin remains
associated with
281
sedimentable
membranes
Vol. 182, No. 1, 1992
(11,12).
However,
ciation (12) be
of
ce
of
most
entrapment
to t h e
rabbit
binding
A) at
that
association
neutral
pH.
D with
of c a t h e p s i n et al.
(I0). D
the
folding,
the
We
have
in U 9 3 7
membranes We
have
optimum
(at l e a s t
no
of the
at p H 5
cells
in U 9 3 7
D pre-
and
thus
supporting
the
is r e l a t e d
that has been been
(12).
in t h e p r e s e n -
apparatus data
not
cannot
of c a t h e p s i n
Golgi
role
we will
may
assist
able
cells
o n e of t h e
carbohydrate
tion
the determination
occurs
The
asso-
reversible
membranes
to o c c u r
binding
to
is
the
to an
described to
detect
using
condi-
(i0).
and maturation
its
shown
to m e m b r a n e s
60 k D a g l y c o p e p t i d e from
membranes
aggregates.
the c r o s s - l i n k i n g
segregation of
cells
cells,
the perforated
has b e e n
proximally
of c a t h e p s i n
of D i m e n t
The
U937
macrophages
reactions
with of t h e
precursors
fibroblastic with
60 kDa g l y c o p e p t i d e
brefeldin probably
tions
D precursor
in c u l t u r e d
possibility
in
by
of t h e s e
cursor
in m o u s e
the association
explained
However,
at least
cathepsin
so t h a t
binding
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of h u m a n attempt
cathepsin
to
of t h e N - t e r m i n a l
isolate amino
large
processing,
D. F o r t h e enough
acid
number
through
of to
elucida-
material
for
sequence.
ACKNOWLEDGMENTS
The authors t h a n k Drs. U s k o k o v i c , Takatsuki, Scangos and Schmidt for generously providing calcitriol, brefeldin A and deoxynojirimycin, Ms. V. F r e i m a n i s f o r t y p i n g a n d Dr. D . B . J o n e s for r e a d i n g t h e m a n u s c r i p t , Die Deutsche Forschungsgemeinschaft and F o n d s d e r C h e m i s c h e n I n d u s t r i e for s u p p o r t i n g t h i s work. REFERENCES
1. 2. 3. 4. -5. 6. 7. 8. 9. i0. ii. 12. 13.
yon Figura, K., and Hasilik, A. (1986) Ann. Rev. Biochem., 55, 167-193. Kornfeld, S. (1987) FASEB J., i, 462-468. Lemansky, P., Gieselmann, V., Hasilik, A., and yon Figura, K., (1984) J. Biol. Chem., 259, 10129-10135. Redecker, B., Horst, M., and Hasilik, A. (1989) Biochem. J. 262, 843-847. Gr~ssel, S., R61ing, A., and Hasilik, A. (1989) Anal. Biochem. 180, 72-78. stein, M., Braulke, T., Krentler, C., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 937-947. Hasilik, A., and Neufeld, E.F. (1980) J. Biol. Chem. 255, 4937-4945. Ulmer, J.B., and Palade, G.E. (1991) J. Biol. Chem. 266, 9173-9179. Radons, J., Isidoro, C., and Hasilik, A. (1990) Biol. Chem. Hoppe-Seyler 371, 567-573. Diment, S., Leech, M.S., and Stahl, P.D. (1988) J. Biol. Chem. 263, 6901-6907. McIntyre, G.F., and Erickson, A.H. (1991) J. Biol. Chem. 266, 15438-15445. Rijnboutt, S., Aerts, H.M.F.G., Geuze, H.J., Tager, J.M., and Strous, G.J. (1991) J. Biol. Chem. 266, 4862-4868. Guagliardi, L.E., Koppelman, B., Blum, J.S., Marks, M.S., Cresswell, P., and Brodsky, F.M. (1990) Nature 343, 133-139.
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