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

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Human cathepsin D precursor is associated with a 60 kDa glycosylated polypeptide.

Human cathepsin D is synthesized as a 53 kDa precursor. Most of it is segregated into lysosomal compartments and subjected to a proteolytic fragmentat...
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