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Saposin D: A sphingomyelinase activator
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 156, No. 1, 1988
Pages 403-410
October 14, 1988
Saposin Satoshi
D:
A Sphingomyelinase
Morimoto*,
Brian and
*Department School
of
of
Health,
Institutes
Molecular
Martin # , Yasuo
Center
University
of
California
of H e a l t h ,
Neurogenetics Bethesda,
K i s h i m o t o *I
S. O ' B r i e n *
Neurosciences,
Medicine,
La Jolla, #National
M.
John
Activator
for
Maryland
San
Genetics, Diego,
92093
National Unit,
Molecular
California
Institute
Building
I0,
for
Mental
Room
3D16,
20892
Received August 25, 1988
SUMMARY: Saposin D, a n e w l y discovered h e a t - s t a b l e , I0 kDa g l y c o p r o t e i n , was isolated f r o m Gaucher s p l e e n and p u r i f i e d to h o m o g e n e i t y . Chemical s e q u e n c i n g from its a m i n o t e r m i n u s d e m o n s t r a t e d c o l i n e a r i t y b e t w e e n its a m i n o acid s e q u e n c e and the d e d u c e d a m i n o acid s e q u e n c e of the f o u r t h d o m a i n of p r o s a p o s i n , the p r e c u r s o r of s a p o s i n p r o t e i n s . S a p o s i n D s p e c i f i c a l l y stimulates acid s p h i n g o m y e l i n a s e b u t has no significant effect on the other h y d r o l a s e s tested. © 1988 A c a d e m i c
Press,
Inc.
I N T R O D U C T I O N : The l y s o s o m a l h y d r o l y s i s of s p h i n g o l i p i d s is c a t a l y z e d b y the sequential
a c t i o n of acid
hydrolases.
Several
enzymatic hydrolytic
steps
are
s t i m u l a t e d b y small h e a t - s t a b l e proteins w h i c h i n t e r a c t w i t h either the s u b s t r a t e or the e n z y m e or both. Two of the h e a t - s t a b l e proteins have b e e n s t r u c t u r a l l y defined. The f i r s t p r o t e i n , n a m e d s a p o s i n B in this r e p o r t a c t i v a t o r of c e r e b r o s i d e s u l f a t a s e ( i ) , GMI p r o t e i n I (3), and d i s p e r s i n (4),
and p r e v i o u s l y d e s i g n a t e d
an
a c t i v a t o r (2), s p h i n g o l i p i d a c t i v a t o r
s t i m u l a t e s the h y d r o l y s i s of
galactocerebroside
sulfate (sulfatide) b y a r y l s u l f a t a s e A (1,5,6), GMI ganglioside by 5 - g a l a c t o s i d a s e (2,7) and g l o b o t r i a o s y l c e r a m i d e (Gb0se3Cer) b y c(-galactosidase A (8).
I m m u n o l o g i c a l and
genetic e v i d e n c e has d e m o n s t r a t e d that saposin B activates all t h r e e r e a c t i o n s (3,8). In a r e c e n t study, Li et al. (9) showed that this protein has
an e v e n b r o a d e r s u b s t r a t e
specificity t h a n r e p o r t e d p r e v i o u s l y , p r o m o t i n g the h y d r o l y s i s of the a b o v e t h r e e glycosphingolipids, as well as GM2 ganglioside, sulfate esters of l a c t o s y l c e r a m i d e and asialo GM2, globoside, t h r e e n e o l a c t o - t y p e glycolipids and two g l y c e r y l g l y c o l i p i d s . From t h e s e r e s u l t s , Li e t al. (9) c o n c l u d e d t h a t the h y d r o l y s i s of t h e s e lipids is
To W h o m
C o r r e s p o n d e n c e S h o u l d Be A d d r e s s e d . 0006-291X/88 $1.50 403
Copyright © 1988 by Academic Press, Inc. All rights" of reproduction in any form reserved.
Vol. 156, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
p r o m o t e d b y a n o n s p e c i f i c d e t e r g e n t action of s a p o s i n B and called it "nonspecific activator protein". The p r i m a r y structure of saposin B was d e t e r m i n e d b y s e q u e n c i n g of a cDNA encoding it (I0). The physiological significance of saposin B is u n d e r s c o r e d b y the d i s c o v e r y of its a b s e n c e in a v a r i a n t form of m e t a c h r o m a t i c l e u c o d y s t r o p h y (8). The second protein, called saposin C in this p a p e r and p r e v i o u s l y t e r m e d factor P (I I), g l u c o c e r e b r o s i d a s e activator (12), coglucosidase (13), s p h i n g o l i p i d activator p r o t e i n 2 (3), and A1 activator (14), stimulates the h y d r o l y s i s of glucosylceramide b y glucosylceramidase (II-14),
t h a t of g a l a c t o c e r e b r o s i d e b y g a l a c t o s y l c e r a m i d e B-
g a l a c t o s i d a s e (15) and t h a t of s p h i n g o m y e l i n b y s p h i n g o m y e l i n a s e (15).
Unlike
saposin B, s a p o s i n C a p p e a r s to i n t e r a c t w i t h e n z y m e s to e x e r t its stimuJatory effect (16). The p r i m a r y s t r u c t u r e of saposin C was d e t e r m i n e d b y chemical s e q u e n c i n g of its amino acids (17, 18) and b y deducing its sequence from nucleotide sequencing of a cDNA e n c o d i n g p r o s a p o s i n , its p r e c u r s o r
(19). The p h y s i o l o g i c a l s i g n i f i c a n c e of
s a p o s i n C w a s d e m o n s t r a t e d b y d i s c o v e r y of its d e f i c i e n c y in a v a r i a n t form of Gaucher's disease (20). Recently, this l a b o r a t o r y has elucidated the complete n u c l e o t i d e s e q u e n c e of a cDNA coding
for prosaposin, the p r e c u r s o r p r o t e i n of both saposin B and C (19).
P r o s a p o s i n was f o u n d to c o n t a i n 4 saposin domains, two of w h i c h are saposin B and C; t h e s e are f l a n k e d b y two additional d o m a i n s we call saposin A and saposin D. Each s a p o s i n d o m a i n (A,B,C,D) is a p p r o x i m a t e l y 80 a m i n o acid r e s i d u e s in length, has n e a r l y i d e n t i c a l p l a c e m e n t of c y s t e i n e r e s i d u e s , g l y c o s y l a t i o n sites and helical r e g i o n s and is f l a n k e d b y proteolytic cleavage sites, Molecular models indicate t h a t the p r o t e i n s d e r i v e d from each d o m a i n can fold to give rise to a c o n f o r m a t i o n a l l y rigid h y d r o p h o b i c pocket held together by
3
disulfide bridges, We p r e d i c t e d t h a t
proteolytic cleavage of p r o s a p o s i n at each d o m a i n b o u n d a r y should give rise to all 4 saposins. We h a v e r e c e n t l y isolated saposin D, the p r o t e i n arising from d o m a i n 4. Saposin D appears
to s p e c i f i c a l l y s t i m u l a t e s p h i n g o m y e l i n a s e to h y d r o l y z e the
p h o s p h o r y l c h o l i n e m o i e t y of s p h i n g o m y e l i n .
METHODS I s o l a t i o n of S a D o s i n I). T e c h n i q u e s used for the i s o l a t i o n of s a p o s i n D were, in general, similar to those used for the isolation of coglucosidase b y Sano and R a d i n (18). Briefly, 201 g r a m s of spleen from a p a t i e n t w i t h a d u l t t y p e Gaucher's disease was h o m o g e n i z e d w i t h 800 ml of w a t e r and the h o m o g e n a t e was h e a t e d at 100°C for I0 min. The m i x t u r e was t h e n centrifuged at I0,000 x g f o r 15 min. and the s u p e r n a t a n t w a s f r a c t i o n a t e d b y a m m o n i u m sulfate p r e c i p i t a t i o n . The p r e c i p i t a t e s w h i c h a p p e a r e d b e t w e e n 45 and 80% s a t u r a t i o n , w e r e collected and dialyzed. The dialysate was t h e n f r a c t i o n a t e d by column c h r o m a t o g r a p h y on DEAE-cellulose (DE-52, W h a t m a n ) . The c o l u m n w a s e l u t e d w i t h a 0-0.SM NaCI l i n e a r g r a d i e n t in 10 mM sodium p h o s p h a t e buffer, pH 7.0. Fractions positive for s t i m u l a t i o n of h y d r o l y s i s of 4m e t h y l u m b e l l i f e r o n e (4-MU) B-D-glucoside (see b e l o w ) w e r e pooled, dialyzed and Ivophilized. The residue, after dissolving in a small v o l u m e of water, was f r a c t i o n a t e d 404
VoI. 156, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by gel f i l t r a t i o n using S e p h a d e x G-75. The f r a c t i o n s c o n t a i n i n g s t i m u l a t i n g a c t i v i t y for 4-MU g l u c o s i d e h y d r o l y s i s w e r e pooled and f u r t h e r f r a c t i o n a t e d b y HPLC. A Varian m o d el 5000 HPLC e q u i p p e d with a Vydac p r o t e i n C4 column (4.6 mm b y 25 cm) was used and f r a c t i o n s w e r e e l u t e d w i t h a l i n e a r g r a d i e n t of w a t e r c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c acid and a c e t o n i t r i l e - w a t e r (4:1) c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c acid. Peaks w e r e d e t e c t e d by m e a s u r i n g a b s o r b a n c e at 220 nm. A typical c h r o m a t o g r a m is s h o w n in Fig. I. The e f f l u e n t comprising t h e saposin D peak w h i c h e l u t e d l at er t h a n saposin B and C ( s h o w n in Fig. I) was collected and e v a p o r a t e d to dryness. .Immunological Procedures: A n t i b o d i e s ag ai n st s a p o s i n D w e r e p r e p a r e d in a r a b b i t as d e s c r i b e d by Crowle (21) and p u r i f i e d w i t h an a f f i n i t y c o l u m n w h i c h c o n t a i n e d p u r i f i e d saposin D linked to Affi-Gel 10 A c t i v a t e d A f f i n i t y S u p p o r t (BioRad) as d e s c r i b e d p r e v i o u s l y (10). Rabbit a n t i b o d i e s ag ai n st sap o si n B and C w e r e p r o v i d e d by Dr. A r v a n F l u h a r t y and Dr. David W en g er , r e s p e c t i v e l y . Dot and W e s t e r n blots w i t h t h e s e antibodies w e r e p e r f o r m e d as d e s c r i b e d pt'eviously (10). A~ys for Lvsosomal Enzyme Activities; Sphingomyelinase activity was assayed as d e s c r i b e d by Wenger (22). [ M e t h y l - 1 4 C ] s p h i n g o m y e l i n o b t a i n e d f r o m Dr. D. A. W e n g e r or f r o m NEN/DuPont, w a s used as s u b s t r a t e . H u m a n p l a c e n t a l s p h i n g o m y e l i n a s e p u r c h a s e d f r o m Sigma Chemical Co. or a Triton X-100 e x t r a c t of a p a r t i c u l a t e f r a c t i o n of h u m a n liver, as d e s c r i b e d by Radin and D e t e n t (23) w as used as an e n z y m e source. Galactoceramide 5 - g a l a c t o s i d a s e and f i - g l u c o s y l c e r a m i d a s e w e r e assayed as d e s c r i b e d b y W e n g e r et a i . ( 1 5 ) . [G-3H]Galactosylceramide w a s p r e p a r e d as d e s c r i b e d p r e v i o u s l y (24). [G-3H]Glucosylceramide w a s a gift f r o m Dr. D. A. W e n g e r . H u m a n s p l e e n w a s h o m o g e n i z e d w i t h two v o l u m e s of w a t e r and c e n t r i f u g e d at 12,500 x g for I0 m i n and t h e s u p e r n a t a n t o b t a i n e d w a s used as an e n z y m e s o u r c e of g a l a c t o s y l c e r a m i d e ~ - g a l a c t o s i d a s e . The s a m e h u m a n l i v e r p r e p a r a t i o n used for s p h i n g o m y e l i n a s e a c t i v i t y w a s used as e n z y m e s o u r c e for f i - g l u c o s y l c e r a m i d a s e assay. GMI g a n g l i o s i d e fJ-galactosidase a c t i v i t y w a s a s s a y e d as d e s c r i b e d p r e v i o u s l y (25). The s u b s t r a t e [ g a l a c t o s e - 3 H ] g a n g l i o s i d e G MI w h i c h w a s s y n t h e s i z e d as d e s c r i b e d (26) and p u r i f i e d h u m a n liver acid f~-galactosidase w e r e p r o v i d e d b y Dr. Yoshimi Y a m a m o t o of this la b o r a t o r y . The h y d r o l y s i s of 4-MU B - D - g l u c o s i d e or fJ-Dgalactoside was p e r f o r m e d as described p r e v i o u s l y (27). O t h e r A n a l y t i c a l M e t h o d s : P e p t i d e s e q u e n c i n g w as a c c o m p l i s h e d using an Applied Biosystems model 470A gas-phase s e q u e n c e r e q u i p p e d w i t h a m o d e l 120A online PTH a n a l y z e r using s t a n d a r d p r o c e d u r e s s u p p l i e d by the m a n u f a c t u r e r . P r o t e i n c o n c e n t r a t i o n and c a r b o h y d r a t e c o n t e n t w e r e d e t e r m i n e d by the m e t h o d of L o w r y e t al. (28) and b y th e phenol sulfuric acid method (29).
RESULTS Isolation from
Gaucher
and spleen
Characterization by
taking
of
advantage
Sa•osin
D. S a p o s i n
of its
heat-stability,
D was
isolated
acidity,
small
m o l e c u l a r size, and h y d r o p h o b i c i t y . Since saposin B and C w e r e also p r e s e n t in t h e DEAE c o l u m n f r a c t i o n s c o n t a i n i n g saposin D its f i n al p u r i f i c a t i o n w a s a c h i e v e d b y p r e p a r a t i v e h y d r o p h o b i c HPLC. The yield of pure s a p o s i n D w a s
16.1 mg f r o m 201 g of
Gaucher s p l e e n . S a p o s i n D w a s e l u t e d w i t h a h i g h e r a c e t o n i t r i l e p e r c e n t a g e t h a n saposin B or C i n d i c a t i v e of its g r e a t e r h y d r o p h o b i c i t y (Fig. I). Saposin D c o n t a i n s a single g l y c o s y l a t i o n site (19) and was f o u n d to co n t ai n 16% c a r b o h y d r a t e b y p h e n o l s u l f u r i c acid analysis.
P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in SDS (Fig. 2) g a v e a
s u b u n i t m o l e c u l a r w e i g h t of a p p r o x i m a t e l y I0 kDa for saposin D similar to saposin B and C. A n t i b o d i e s raised against saposin D did not cross r eact w i t h saposin B or C (Fig. 2). C o n v e r s e l y , a n t i b o d i e s against saposin B or C did n o t cross r e a c t w i t h s a p o s i n D 405
VoI. 156, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
o. re
B
Q
~
,'o
2'o
~'o
C
4'o
5'o
7'0 ,.i.'.
go
(~
1
2
3
4
1
2
3
4
Figure I. HPLC of SaDosin D. Purified saposin D was analyzed b y HPLC as described in Methods. Under the same conditions, saposin B and C a p p e a r w i t h r e t e n t i o n times as indicated b y arrows. Yi~ure 2. S D S - P o l y a c r v l a m i d e Gel E 1 e c t r o D h o r e s i s a n d I m m u n o b l o t t i n ~ of SaDosin D. The p u r i f i e d saposin D (8.6 ~tg, lane 2 and 6) w a s e l e c t r o p h o r e s e d together w i t h saposin C (7.6 }~g, lane 3 and 7) and saposin B (13.6 ~tg, lane 4 and 8) on 17.5% p o l y a c r y l a m i d e slab gels containing SDS. Lanes I and 5 c o n t a i n e d a protein s t a n d a r d m i x t u r e (Sigma Chemical Co.). The lowest protein m a r k e r is ¢ x - l a c t a l b u m i n (14,200 Da). Section A w a s s t a i n e d w i t h Coomassie b r i l l i a n t b l u e and B w a s i m m u n o b l o t t e d w i t h a n t i - s a p o s i n D a n t i s e r u m as described in Methods.
(data not shown). revealed
Chemical sequencing
of t h e N - t e r m i n a l
amino
a c i d s of s a p o s i n D
t h e f i r s t 19 a m i n o a c i d s to b e i d e n t i c a l to t h e p o l y p e p t i d e
predicted
to a r i s e
f r o m p r o t e o l y s i s of d o m a i n 4 of p r o s a p o s i n ( a m i n o acids 3 9 2 to 4 7 4 ) (19). Specificity
of
Sphingomyelinase
Stimulation
p r e s e n c e of T r i t o n X - 1 0 0 , s p h i n g o m y e l i n a s e
by
Saposin
activity was stimulated
D, I n
the
100% b y as l i t t l e
as 2 ~Ig (less t h a n 1 }~M) of s a p o s i n D as s h o w n i n Fig. 3. S t i m u l a t i o n w a s n o t i n c r e a s e d when
the
amount
concentration.
of s a p o s i n
Stimulation
D was
was
increased
indicating
near
n o t l i k e l y d u e to a n o n s p e c i f i c
saturation detergent
at this
nature
of
s a p o s i n D b e c a u s e t h e a s s a y m i x t u r e c o n t a i n e d s u f f i c i e n t T r i t o n X - 1 0 0 to s o l u b i l i z e t h e sphingomyelin
present
glucosylceramidase
in the
activity
assay.
(16,
In addition,
30)
(17,18,19,3 i) did not possess stimulatory
and
has
saposin
a structure
C, w h i c h similar
the
presence
galactosidase enhanced
of
~-galactoside,
6-galactosidase
I). T h e h y d r o l y s i s
saposin D was
hydrolysis
pyrophosphate, glucuronide,
galactosylceramide
activity (Table
when
stimulate
saposin
D
a c t i v i t y 3 - a n d 5-
H o w e v e r , t h e a c t i v i t y o b t a i n e d w i t h 5 4 llg w a s o n l y 10% of t h a t i n
of T r i t o n X - 1 0 0 . S a p o s i n D d i d n o t s t i m u l a t e
a c t i v i t y (Fig. 3),
to
13-
a c t i v i t y in t h e a s s a y . W i t h a d d i t i o n of T r i t o n
X - 1 0 0 , 2 7 ~tg a n d 5 4 }.tg of S a p o s i n D i n c r e a s e d t h e s p h i n g o m y e l i n a s e fold, r e s p e c t i v e l y .
stimulates
present
the
other
ganglioside
of 4 - M U 8 - D - g l u c o s i d e
(also shown in Table 4-MU
•-glucosylceramidase
a c t i v i t y or G M I
derivative
was
13-
slightly
I). S a p o s i n D d i d n o t substrates
including
13-galactoside, a - g l u c o s i d e , c x - m a n n o s i d e , 13-fucoside, 13=
and •-N-acetylglucosamide
(data not shown), 406
Vol. 156, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0-
[]
0.8 = 0.7-
0.60.5,
o
"o 0.~I E c
0.3'
0.2. 0.1!
©
D-)l~ug Saposin
)'5 C or
2'0
D
Figure 3, Effect of Sa~osin D on SDhin~omvelinase a n d G 1 u c o c e r e b r o s i d a s e , Various amounts of saposin D w e r e a d d e d to an assay mixture of s p h i n g o m y e l i n a s e and fl-glucosylceramidase activities and its effect was c o m p a r e d w i t h t h a t of saposin C. Sphingomyelinase activity assay mixture contained 20 nmol [methyl-14C] sphingomyelin (830 cpm/nmol), I0 lag Triton X-100, and 50 lamol acetate buffer pH 5.0 in 200 )II. Crude s p h i n g o m y e l i n a s e p r e p a r e d f r o m h u m a n p l a c e n t a (Sigma Chem. Co.) was used as enzyme source. Incubation was conducted at 37°C for I hr. 13-Glucosylceramidase a s s a y m i x t u r e c o n t a i n e d 5.25 n m o l to [ g l u c o - 3 H ) g l u c o c e r e b r o s i d e (1358 c p m / n m o l ) and 20 lamol c i t r a t e - p h o s p h a t e b u f f e r pH 4.2 in 200 lal. Human liver particulate preparation solubilized by 0.1% Triton X-100 was used as e n z y m e source. The activities for s p h i n g o m y e l i n a s e w i t h saposin D is s h o w n by open squares ( [ ] ) and t h a t w i t h saposin C is shown by closed squares ( • ) . The activity for 13-glucosylceramidase w i t h saposin D is e x p r e s s e d by o p e n circle ( O ) and t h a t w i t h s a p o s i n C is e x p r e s s e d by closed circle ( 11 ). No f u r t h e r i n c r e a s e of ~glucosylceramidase activity was observed by increasing saposins up to 35 lag each.
DISCUSSION In a c c o r d a n c e w i t h prosaposin generates i s o l a t i o n of a t h i r d
our recent
prediction that
the
proteolytic
4 s a p o s i n p r o t e i n s of s i m i l a r s t r u c t u r e ( 1 9 ) w e s a p o s i n p r o t e i n , s a p o s i n D, w h i c h
stimulates
processing
of
report here the the hydrolysis
of
s p h i n g o m y e l i n . P r o o f t h a t s a p o s i n D a r i s e s f r o m d o m a i n 4 of p r o s a p o s i n w a s o b t a i n e d by demonstrating and
the
addition,
c o l i n e a r i t y b e t w e e n its f i r s t 19 c h e m i c a l l y d e t e r m i n e d
deduced saposin
sequence D has
obtained
the
correct
from
domain
predicted
4 of t h e
molecular
a m i n o acids
prosaposin
weight
(10
cDNA. I n kDa)
p r o t e o l y t i c p r o c e s s i n g of d o m a i n 4. W h i l e t h i s w o r k w a s in p r o g r e s s F u r s t e t al. isolated the same protein from Gaucher spleen termed
component
from (31)
C; its a m i n o acid
s e q u e n c e is i d e n t i c a l t o s a p o s i n D e x c e p t f o r t h e a b s e n c e of t h e f i r s t t w o N - t e r m i n a l amino
acids which
may
have
been
removed
by
peptidase
a c t i o n . No a c t i v a t i n g
p r o p e r t i e s w e r e r e p o r t e d f o r c o m p o n e n t C. Saposin D has a remarkably
high sp e c i f i c i t y in s t i m u l a t i n g s p h i n g o m y e l i n a s e
a c t i v i t y . A c t i v a t i o n of s p h i n g o m y e l i n a s e a c t i v i t y b y s a p o s i n C h a s also b e e n r e p o r t e d 407
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I. Effect of Saposin D on GM 1 G a n g l i o s i d e g-Galactosidase, Galactocerebroside B-Galactosidase a n d 4 - M e t h y l u m b e l l i f e r y l Glucoside l~-G1ucosidase
Saposin
=,,
(~g)
nmol hydrolyzed/hr Saposin + Saposin
GMI G a n g l i o s i d e ~-galactosidase
B (42) D (45)
0 0
0.44 0.00
Galactosylceramidase
C (8) (16) (24)
0.41 0.41 0.41
0.57 1.24 1,48
D (14) (54) (95)
0.41 0.41 0.41
0.34 0.44 0.44
C (4) ( I 0) (25)
3.70 3.70 3.7O
9.00 t3.80 13.80
D (4) (10) (25)
3.70 3.70 3.70
5.50 5.00 5.00
4 - M e t h y l u m b e l l i f e r y l glucoside B - g l u c o s i d ase
(15); b u t in our h a n d s s a p o s i n C failed to s t i m u l a t e s p h i n g o m y e l i n a s e a c t i v i t y . Christomanou and her colleagues (20) purified two proteins, n a m e d A I and A 2, which stimulated
s p h i n g o m y e l i n a s e activity; t h e i r
A I p r o t e i n was f o u n d b y c h e m i c a l
s e q u e n c i n g to be i d e n t i c a l to saposin C (17). It is possible t h a t our HPLC-purified s a p o s i n C lost its s p h i n g o m y e l i n a s e a c t i v a t i n g p r o p e r t y
w h i l e r e t a i n i n g its tS-
g l u c o s y l c e r a m i d a s e s t i m u l a t i n g p r o p e r t y . A l t e r n a t i v e l y , p r e v i o u s p r e p a r a t i o n s of saposin C m a y have b e e n c o n t a m i n a t e d w i t h saposin D since their properties are v e r y s i m i l a r and e x t r e m e l y s m a l l a m o u n t s of s a p o s i n D are r e q u i r e d for a c t i v a t i o n . A n o t h e r e x p l a n a t i o n is the d e t e r g e n t - l i k e p r o p e r t y of s a p o s i n C w h i c h h e l p e d to solubilize s p h i n g o m y e l i n in the absence of Triton X-100. Prior to the discovery that saposin 13 and C are both d e r i v e d from the same p r e c u r s o r p r o t e i n (19), t h e s e proteins w e r e n a m e d n o n s y s t e m a t i c a l l y . The isolation of
saposin
D reported
nomenclature
for
the
here saposin
has
prompted
proteins.
As
us
to
propose
mentioned
the
a new
systematic
precursor
protein
( p r o s a p o s i n ) c o n t a i n s 4 d o m a i n s comprised of similar 80 a m i n o acid m o d u l a r units w h i c h are r e l e a s e d
u p o n proteolysis. We p r o p o s e t h a t t h o s e p r o t e i n s w h i c h are
derived from the first, second, third and f o u r t h domains be n a m e d saposin A, 13, C and D. Of the four, s a p o s i n A has y e t to be isolated. Partial proteolysis of prosaposin also 408
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
y i e l d s i n t e r m e d i a t e c l e a v a g e p r o d u c t s and t h e s e h a v e b e e n d e t e c t e d in most h u m a n t i s s u e s (19). We p r o p o s e t h a t p r o t e i n s p o s s e s s i n g two s a p o s i n d o m a i n s w i t h s u b u n i t molecular w e i g h t s of 2 5 - 3 0 kDa be called d i s a p o s i n A, B and C; containing d o m a i n s I and 2, 2 and 3, and 3 and 4 r e s p e c t i v e l y . S i m i l a r l y , c l e a v a g e p r o d u c t s c o n t a i n i n g t h r e e s a p o s i n d o m a i n s w i t h s u b u n i t m o l e c u l a r w e i g h t s of a p p r o x i m a t e l y 48 and 43 kDa, be n a m e d t r i s a p o s i n A and B; containing d o m a i n s 1,2 and 3 and d o m a i n s 2, 3, 4 r e s p e c t i v e l y . I n l i n e w i t h this proposal, w e h a v e r e c e n t l y i s o l a t e d a g l y c o p r o t e i n from Gaucher s p l e e n w h i c h a p p e a r s to be d i s a p o s i n A.
ACKNOWLEDGEMENTS We a r e g r a t e f u l to Dr. David W e n g e r for p r o v i d i n g us w i t h a n t i b o d i e s a g a i n s t saposin C and r a d i o a c t i v e sphingolipid s u b s t r a t e s , to Dr. A r y a n F l u h a r t y for giving us a n t i b o d i e s a g a i n s t s a p o s i n B,
to Dr. Yoshimi Y a m a m o t o for p u r i f i e d f}-galactosidase
and
GM I, and to Dr. N o r m a n R a d i n for s h o w i n g us an
radioactive
unpublished
ganglioside
manuscript.
We
also t h a n k
Susan
O'Brien for
her
assistance
in
p r e p a r a t i o n of this m a n u s c r i p t . This s t u d y w a s s u p p o r t e d in p a r t b y NIH r e s e a r c h grants NS-13559 (YK) and HD-18983 and NS-08682 (JOB).
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. I I. 12. 13. 14. 15. 16. 17. 18. 19.
Fisher, G. and Jatzkewitz, H. (1975) H o p p e - S e y l e r ' s Z. Physiol. Chem. 356, 6 0 5 613. Li, S.-C. and Li, Y.-T. (1976) J. Biol. Chem. 25 I, 1159-1163. W e n g e r , D.A. and Inui, K. (1984) In The Molecular Basis of L y s o s o m a l S t o r a g e Disorders (R. O. B r a d y and J.A. Barranger, Eds.) pp. 61-78. A c a d e m i c Press, New York. Li, S.-C. and Li, Y.-T. (I 986) NATO ASI Ser. A. Life Sci. 1 1 6 , 3 0 7 - 3 1 4 . Mehl, E. and Jatzkewitz, H. (1964) H o p p e - S e y l e r ' s Z. Physiol. Chem. 339, 2 6 0 - 2 7 6 . Fischer, G. and Jatzkewitz, H. (1978) Biochim. Biophys. Acta 528, 69-76. Li, S.-C., Wan, C.C., Mazzotta, M.Y. and Li, Y.-T. (1974) C a r b o h y d r . Res. 34, 189193. Li, S.-C., Kihara, H. S e r i z a w a , S., Li, Y.-T., F l u h a r t y , A.L., M a y e s , J.S., and Shapiro, L.J.(1985) J. Biol. Chem. 260, 1867-1871. Li, S.-C., Sonnino, S., Tettamanti, G., and Li, Y.-T. (1988) J. Biol. Chem. 263, 6 5 8 8 6591. Dewji, N., W e n g e r , D., Fujibayashi, S., Donoviel, M., Esch, F., Hill, F., O'Brien, J.S. (1986) Biochem. Biophys. Res. Comm. 134, 9 8 9 - 9 9 4 . Ho. M.W. and O'Brien, J.S. (1971) Proc. Natl. Acad. Sci. USA 68, 2 8 1 0 - 2 8 1 3 . P e t e r s , S.P., Coffee, C.J., Glew, R.H., K u h l e n s c h m i d t , M.S., Rosenfeld, L., Lee, Y.C. (1977) J. Biol. Chem. 252, 563-573. Berent, S.L. and Radin, N.S. (1981) Arch. Biochem. Biophys. 208, 2 4 8 - 2 6 0 . C h r i s t o m a n o u , H. and Kleinschmidt, T. ( 1 9 8 5 ) Biol. Chem. H o p p e - S e y l e r 366, 245-256. W e n g e r , B.A., Sattler, M., Roth, S. (1982) Biochim. Biophys. Acta 712, 6 3 9 - 6 4 9 . Radin, N.S. ( 1 9 8 4 ) In Molecular Basis of L y s o s o m a l S t o r a g e D i s o r d e r s (R.O. B r a d y and J.A. B a r r a n g e r Eds.), pp. 9 3 - I 12. A c a d e m i c Press, New York. Kleinschmidt, T., Christomanou, H., and B r a u n i t z e r G. (1987) Biol. Chem. H o p p e Seyler 368, 1571-1578. Sano A., and Radin, N.S. (1988) FESEB J. 2: A1779. O'Brien, J.S., Kretz, K.A., Dewji, N.N., W e n g e r , B.A., Esch, F. and F l u h a r t y , A.L. (1988) Science, in press. 409
Vol. 156, No. 1, 1988
20. 21. 22. 23. 24 25. 26. 27. 28. 29. 30. 31.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Christomanou, H., Aignesberger, A. and Linke, R.P. (1986) Biol. Chem. HoppeSeyler 367, 879-890. Crowle, A.J. (1972) Immunodiffusion, 2rid Edition, pp. 78-96. Academic Press, New York. Wenger, D.A. ( 1 9 7 7 ) In Practical E n z y m o l o g y of the Sphingolipidoses (R.H. Glew and S.P. Peters, Eds.) pp. 39-70. Alan R. Liss Inc., New York. Radin, N.S. and Berent, S.L. (1982) Meth. Enzymol. 83, 5 9 6 % 0 3 . Hajra, A,K., Bowen, D.M., Kishimoto, Y. and Radin N.S. (1966) J. Lipid Res. 7, 379386. Norden, A.G.W. and O'Brien, J.S. (1973) Arch. 13iochem. Biophys. 159, 383-392. Suzuki, Y. and Suzuki, K. (1972) J. Lipid Res. 13, 687-690. Ho, M.W. and O'Brien, J.S. (1973) Biochem. J. 131, 173-176. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275. Ashwell, G. (1966) Meth. Enzymol. 8, 93-95. Glew, R.H., 13asu, A., LaMarco, K.L., and Prence, E.M. (1988) Lab. Invest. 58, 5-25. Furst, W., Machleidt, W., and Sandhoff, K. (1988) Biol. Chem. Hoppe-Seyler 369, 317-328.
410
Vol. 156, No. 1, 1988
Pages 403-410
October 14, 1988
Saposin Satoshi
D:
A Sphingomyelinase
Morimoto*,
Brian and
*Department School
of
of
Health,
Institutes
Molecular
Martin # , Yasuo
Center
University
of
California
of H e a l t h ,
Neurogenetics Bethesda,
K i s h i m o t o *I
S. O ' B r i e n *
Neurosciences,
Medicine,
La Jolla, #National
M.
John
Activator
for
Maryland
San
Genetics, Diego,
92093
National Unit,
Molecular
California
Institute
Building
I0,
for
Mental
Room
3D16,
20892
Received August 25, 1988
SUMMARY: Saposin D, a n e w l y discovered h e a t - s t a b l e , I0 kDa g l y c o p r o t e i n , was isolated f r o m Gaucher s p l e e n and p u r i f i e d to h o m o g e n e i t y . Chemical s e q u e n c i n g from its a m i n o t e r m i n u s d e m o n s t r a t e d c o l i n e a r i t y b e t w e e n its a m i n o acid s e q u e n c e and the d e d u c e d a m i n o acid s e q u e n c e of the f o u r t h d o m a i n of p r o s a p o s i n , the p r e c u r s o r of s a p o s i n p r o t e i n s . S a p o s i n D s p e c i f i c a l l y stimulates acid s p h i n g o m y e l i n a s e b u t has no significant effect on the other h y d r o l a s e s tested. © 1988 A c a d e m i c
Press,
Inc.
I N T R O D U C T I O N : The l y s o s o m a l h y d r o l y s i s of s p h i n g o l i p i d s is c a t a l y z e d b y the sequential
a c t i o n of acid
hydrolases.
Several
enzymatic hydrolytic
steps
are
s t i m u l a t e d b y small h e a t - s t a b l e proteins w h i c h i n t e r a c t w i t h either the s u b s t r a t e or the e n z y m e or both. Two of the h e a t - s t a b l e proteins have b e e n s t r u c t u r a l l y defined. The f i r s t p r o t e i n , n a m e d s a p o s i n B in this r e p o r t a c t i v a t o r of c e r e b r o s i d e s u l f a t a s e ( i ) , GMI p r o t e i n I (3), and d i s p e r s i n (4),
and p r e v i o u s l y d e s i g n a t e d
an
a c t i v a t o r (2), s p h i n g o l i p i d a c t i v a t o r
s t i m u l a t e s the h y d r o l y s i s of
galactocerebroside
sulfate (sulfatide) b y a r y l s u l f a t a s e A (1,5,6), GMI ganglioside by 5 - g a l a c t o s i d a s e (2,7) and g l o b o t r i a o s y l c e r a m i d e (Gb0se3Cer) b y c(-galactosidase A (8).
I m m u n o l o g i c a l and
genetic e v i d e n c e has d e m o n s t r a t e d that saposin B activates all t h r e e r e a c t i o n s (3,8). In a r e c e n t study, Li et al. (9) showed that this protein has
an e v e n b r o a d e r s u b s t r a t e
specificity t h a n r e p o r t e d p r e v i o u s l y , p r o m o t i n g the h y d r o l y s i s of the a b o v e t h r e e glycosphingolipids, as well as GM2 ganglioside, sulfate esters of l a c t o s y l c e r a m i d e and asialo GM2, globoside, t h r e e n e o l a c t o - t y p e glycolipids and two g l y c e r y l g l y c o l i p i d s . From t h e s e r e s u l t s , Li e t al. (9) c o n c l u d e d t h a t the h y d r o l y s i s of t h e s e lipids is
To W h o m
C o r r e s p o n d e n c e S h o u l d Be A d d r e s s e d . 0006-291X/88 $1.50 403
Copyright © 1988 by Academic Press, Inc. All rights" of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
p r o m o t e d b y a n o n s p e c i f i c d e t e r g e n t action of s a p o s i n B and called it "nonspecific activator protein". The p r i m a r y structure of saposin B was d e t e r m i n e d b y s e q u e n c i n g of a cDNA encoding it (I0). The physiological significance of saposin B is u n d e r s c o r e d b y the d i s c o v e r y of its a b s e n c e in a v a r i a n t form of m e t a c h r o m a t i c l e u c o d y s t r o p h y (8). The second protein, called saposin C in this p a p e r and p r e v i o u s l y t e r m e d factor P (I I), g l u c o c e r e b r o s i d a s e activator (12), coglucosidase (13), s p h i n g o l i p i d activator p r o t e i n 2 (3), and A1 activator (14), stimulates the h y d r o l y s i s of glucosylceramide b y glucosylceramidase (II-14),
t h a t of g a l a c t o c e r e b r o s i d e b y g a l a c t o s y l c e r a m i d e B-
g a l a c t o s i d a s e (15) and t h a t of s p h i n g o m y e l i n b y s p h i n g o m y e l i n a s e (15).
Unlike
saposin B, s a p o s i n C a p p e a r s to i n t e r a c t w i t h e n z y m e s to e x e r t its stimuJatory effect (16). The p r i m a r y s t r u c t u r e of saposin C was d e t e r m i n e d b y chemical s e q u e n c i n g of its amino acids (17, 18) and b y deducing its sequence from nucleotide sequencing of a cDNA e n c o d i n g p r o s a p o s i n , its p r e c u r s o r
(19). The p h y s i o l o g i c a l s i g n i f i c a n c e of
s a p o s i n C w a s d e m o n s t r a t e d b y d i s c o v e r y of its d e f i c i e n c y in a v a r i a n t form of Gaucher's disease (20). Recently, this l a b o r a t o r y has elucidated the complete n u c l e o t i d e s e q u e n c e of a cDNA coding
for prosaposin, the p r e c u r s o r p r o t e i n of both saposin B and C (19).
P r o s a p o s i n was f o u n d to c o n t a i n 4 saposin domains, two of w h i c h are saposin B and C; t h e s e are f l a n k e d b y two additional d o m a i n s we call saposin A and saposin D. Each s a p o s i n d o m a i n (A,B,C,D) is a p p r o x i m a t e l y 80 a m i n o acid r e s i d u e s in length, has n e a r l y i d e n t i c a l p l a c e m e n t of c y s t e i n e r e s i d u e s , g l y c o s y l a t i o n sites and helical r e g i o n s and is f l a n k e d b y proteolytic cleavage sites, Molecular models indicate t h a t the p r o t e i n s d e r i v e d from each d o m a i n can fold to give rise to a c o n f o r m a t i o n a l l y rigid h y d r o p h o b i c pocket held together by
3
disulfide bridges, We p r e d i c t e d t h a t
proteolytic cleavage of p r o s a p o s i n at each d o m a i n b o u n d a r y should give rise to all 4 saposins. We h a v e r e c e n t l y isolated saposin D, the p r o t e i n arising from d o m a i n 4. Saposin D appears
to s p e c i f i c a l l y s t i m u l a t e s p h i n g o m y e l i n a s e to h y d r o l y z e the
p h o s p h o r y l c h o l i n e m o i e t y of s p h i n g o m y e l i n .
METHODS I s o l a t i o n of S a D o s i n I). T e c h n i q u e s used for the i s o l a t i o n of s a p o s i n D were, in general, similar to those used for the isolation of coglucosidase b y Sano and R a d i n (18). Briefly, 201 g r a m s of spleen from a p a t i e n t w i t h a d u l t t y p e Gaucher's disease was h o m o g e n i z e d w i t h 800 ml of w a t e r and the h o m o g e n a t e was h e a t e d at 100°C for I0 min. The m i x t u r e was t h e n centrifuged at I0,000 x g f o r 15 min. and the s u p e r n a t a n t w a s f r a c t i o n a t e d b y a m m o n i u m sulfate p r e c i p i t a t i o n . The p r e c i p i t a t e s w h i c h a p p e a r e d b e t w e e n 45 and 80% s a t u r a t i o n , w e r e collected and dialyzed. The dialysate was t h e n f r a c t i o n a t e d by column c h r o m a t o g r a p h y on DEAE-cellulose (DE-52, W h a t m a n ) . The c o l u m n w a s e l u t e d w i t h a 0-0.SM NaCI l i n e a r g r a d i e n t in 10 mM sodium p h o s p h a t e buffer, pH 7.0. Fractions positive for s t i m u l a t i o n of h y d r o l y s i s of 4m e t h y l u m b e l l i f e r o n e (4-MU) B-D-glucoside (see b e l o w ) w e r e pooled, dialyzed and Ivophilized. The residue, after dissolving in a small v o l u m e of water, was f r a c t i o n a t e d 404
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by gel f i l t r a t i o n using S e p h a d e x G-75. The f r a c t i o n s c o n t a i n i n g s t i m u l a t i n g a c t i v i t y for 4-MU g l u c o s i d e h y d r o l y s i s w e r e pooled and f u r t h e r f r a c t i o n a t e d b y HPLC. A Varian m o d el 5000 HPLC e q u i p p e d with a Vydac p r o t e i n C4 column (4.6 mm b y 25 cm) was used and f r a c t i o n s w e r e e l u t e d w i t h a l i n e a r g r a d i e n t of w a t e r c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c acid and a c e t o n i t r i l e - w a t e r (4:1) c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c acid. Peaks w e r e d e t e c t e d by m e a s u r i n g a b s o r b a n c e at 220 nm. A typical c h r o m a t o g r a m is s h o w n in Fig. I. The e f f l u e n t comprising t h e saposin D peak w h i c h e l u t e d l at er t h a n saposin B and C ( s h o w n in Fig. I) was collected and e v a p o r a t e d to dryness. .Immunological Procedures: A n t i b o d i e s ag ai n st s a p o s i n D w e r e p r e p a r e d in a r a b b i t as d e s c r i b e d by Crowle (21) and p u r i f i e d w i t h an a f f i n i t y c o l u m n w h i c h c o n t a i n e d p u r i f i e d saposin D linked to Affi-Gel 10 A c t i v a t e d A f f i n i t y S u p p o r t (BioRad) as d e s c r i b e d p r e v i o u s l y (10). Rabbit a n t i b o d i e s ag ai n st sap o si n B and C w e r e p r o v i d e d by Dr. A r v a n F l u h a r t y and Dr. David W en g er , r e s p e c t i v e l y . Dot and W e s t e r n blots w i t h t h e s e antibodies w e r e p e r f o r m e d as d e s c r i b e d pt'eviously (10). A~ys for Lvsosomal Enzyme Activities; Sphingomyelinase activity was assayed as d e s c r i b e d by Wenger (22). [ M e t h y l - 1 4 C ] s p h i n g o m y e l i n o b t a i n e d f r o m Dr. D. A. W e n g e r or f r o m NEN/DuPont, w a s used as s u b s t r a t e . H u m a n p l a c e n t a l s p h i n g o m y e l i n a s e p u r c h a s e d f r o m Sigma Chemical Co. or a Triton X-100 e x t r a c t of a p a r t i c u l a t e f r a c t i o n of h u m a n liver, as d e s c r i b e d by Radin and D e t e n t (23) w as used as an e n z y m e source. Galactoceramide 5 - g a l a c t o s i d a s e and f i - g l u c o s y l c e r a m i d a s e w e r e assayed as d e s c r i b e d b y W e n g e r et a i . ( 1 5 ) . [G-3H]Galactosylceramide w a s p r e p a r e d as d e s c r i b e d p r e v i o u s l y (24). [G-3H]Glucosylceramide w a s a gift f r o m Dr. D. A. W e n g e r . H u m a n s p l e e n w a s h o m o g e n i z e d w i t h two v o l u m e s of w a t e r and c e n t r i f u g e d at 12,500 x g for I0 m i n and t h e s u p e r n a t a n t o b t a i n e d w a s used as an e n z y m e s o u r c e of g a l a c t o s y l c e r a m i d e ~ - g a l a c t o s i d a s e . The s a m e h u m a n l i v e r p r e p a r a t i o n used for s p h i n g o m y e l i n a s e a c t i v i t y w a s used as e n z y m e s o u r c e for f i - g l u c o s y l c e r a m i d a s e assay. GMI g a n g l i o s i d e fJ-galactosidase a c t i v i t y w a s a s s a y e d as d e s c r i b e d p r e v i o u s l y (25). The s u b s t r a t e [ g a l a c t o s e - 3 H ] g a n g l i o s i d e G MI w h i c h w a s s y n t h e s i z e d as d e s c r i b e d (26) and p u r i f i e d h u m a n liver acid f~-galactosidase w e r e p r o v i d e d b y Dr. Yoshimi Y a m a m o t o of this la b o r a t o r y . The h y d r o l y s i s of 4-MU B - D - g l u c o s i d e or fJ-Dgalactoside was p e r f o r m e d as described p r e v i o u s l y (27). O t h e r A n a l y t i c a l M e t h o d s : P e p t i d e s e q u e n c i n g w as a c c o m p l i s h e d using an Applied Biosystems model 470A gas-phase s e q u e n c e r e q u i p p e d w i t h a m o d e l 120A online PTH a n a l y z e r using s t a n d a r d p r o c e d u r e s s u p p l i e d by the m a n u f a c t u r e r . P r o t e i n c o n c e n t r a t i o n and c a r b o h y d r a t e c o n t e n t w e r e d e t e r m i n e d by the m e t h o d of L o w r y e t al. (28) and b y th e phenol sulfuric acid method (29).
RESULTS Isolation from
Gaucher
and spleen
Characterization by
taking
of
advantage
Sa•osin
D. S a p o s i n
of its
heat-stability,
D was
isolated
acidity,
small
m o l e c u l a r size, and h y d r o p h o b i c i t y . Since saposin B and C w e r e also p r e s e n t in t h e DEAE c o l u m n f r a c t i o n s c o n t a i n i n g saposin D its f i n al p u r i f i c a t i o n w a s a c h i e v e d b y p r e p a r a t i v e h y d r o p h o b i c HPLC. The yield of pure s a p o s i n D w a s
16.1 mg f r o m 201 g of
Gaucher s p l e e n . S a p o s i n D w a s e l u t e d w i t h a h i g h e r a c e t o n i t r i l e p e r c e n t a g e t h a n saposin B or C i n d i c a t i v e of its g r e a t e r h y d r o p h o b i c i t y (Fig. I). Saposin D c o n t a i n s a single g l y c o s y l a t i o n site (19) and was f o u n d to co n t ai n 16% c a r b o h y d r a t e b y p h e n o l s u l f u r i c acid analysis.
P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in SDS (Fig. 2) g a v e a
s u b u n i t m o l e c u l a r w e i g h t of a p p r o x i m a t e l y I0 kDa for saposin D similar to saposin B and C. A n t i b o d i e s raised against saposin D did not cross r eact w i t h saposin B or C (Fig. 2). C o n v e r s e l y , a n t i b o d i e s against saposin B or C did n o t cross r e a c t w i t h s a p o s i n D 405
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A
B
o. re
B
Q
~
,'o
2'o
~'o
C
4'o
5'o
7'0 ,.i.'.
go
(~
1
2
3
4
1
2
3
4
Figure I. HPLC of SaDosin D. Purified saposin D was analyzed b y HPLC as described in Methods. Under the same conditions, saposin B and C a p p e a r w i t h r e t e n t i o n times as indicated b y arrows. Yi~ure 2. S D S - P o l y a c r v l a m i d e Gel E 1 e c t r o D h o r e s i s a n d I m m u n o b l o t t i n ~ of SaDosin D. The p u r i f i e d saposin D (8.6 ~tg, lane 2 and 6) w a s e l e c t r o p h o r e s e d together w i t h saposin C (7.6 }~g, lane 3 and 7) and saposin B (13.6 ~tg, lane 4 and 8) on 17.5% p o l y a c r y l a m i d e slab gels containing SDS. Lanes I and 5 c o n t a i n e d a protein s t a n d a r d m i x t u r e (Sigma Chemical Co.). The lowest protein m a r k e r is ¢ x - l a c t a l b u m i n (14,200 Da). Section A w a s s t a i n e d w i t h Coomassie b r i l l i a n t b l u e and B w a s i m m u n o b l o t t e d w i t h a n t i - s a p o s i n D a n t i s e r u m as described in Methods.
(data not shown). revealed
Chemical sequencing
of t h e N - t e r m i n a l
amino
a c i d s of s a p o s i n D
t h e f i r s t 19 a m i n o a c i d s to b e i d e n t i c a l to t h e p o l y p e p t i d e
predicted
to a r i s e
f r o m p r o t e o l y s i s of d o m a i n 4 of p r o s a p o s i n ( a m i n o acids 3 9 2 to 4 7 4 ) (19). Specificity
of
Sphingomyelinase
Stimulation
p r e s e n c e of T r i t o n X - 1 0 0 , s p h i n g o m y e l i n a s e
by
Saposin
activity was stimulated
D, I n
the
100% b y as l i t t l e
as 2 ~Ig (less t h a n 1 }~M) of s a p o s i n D as s h o w n i n Fig. 3. S t i m u l a t i o n w a s n o t i n c r e a s e d when
the
amount
concentration.
of s a p o s i n
Stimulation
D was
was
increased
indicating
near
n o t l i k e l y d u e to a n o n s p e c i f i c
saturation detergent
at this
nature
of
s a p o s i n D b e c a u s e t h e a s s a y m i x t u r e c o n t a i n e d s u f f i c i e n t T r i t o n X - 1 0 0 to s o l u b i l i z e t h e sphingomyelin
present
glucosylceramidase
in the
activity
assay.
(16,
In addition,
30)
(17,18,19,3 i) did not possess stimulatory
and
has
saposin
a structure
C, w h i c h similar
the
presence
galactosidase enhanced
of
~-galactoside,
6-galactosidase
I). T h e h y d r o l y s i s
saposin D was
hydrolysis
pyrophosphate, glucuronide,
galactosylceramide
activity (Table
when
stimulate
saposin
D
a c t i v i t y 3 - a n d 5-
H o w e v e r , t h e a c t i v i t y o b t a i n e d w i t h 5 4 llg w a s o n l y 10% of t h a t i n
of T r i t o n X - 1 0 0 . S a p o s i n D d i d n o t s t i m u l a t e
a c t i v i t y (Fig. 3),
to
13-
a c t i v i t y in t h e a s s a y . W i t h a d d i t i o n of T r i t o n
X - 1 0 0 , 2 7 ~tg a n d 5 4 }.tg of S a p o s i n D i n c r e a s e d t h e s p h i n g o m y e l i n a s e fold, r e s p e c t i v e l y .
stimulates
present
the
other
ganglioside
of 4 - M U 8 - D - g l u c o s i d e
(also shown in Table 4-MU
•-glucosylceramidase
a c t i v i t y or G M I
derivative
was
13-
slightly
I). S a p o s i n D d i d n o t substrates
including
13-galactoside, a - g l u c o s i d e , c x - m a n n o s i d e , 13-fucoside, 13=
and •-N-acetylglucosamide
(data not shown), 406
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0-
[]
0.8 = 0.7-
0.60.5,
o
"o 0.~I E c
0.3'
0.2. 0.1!
©
D-)l~ug Saposin
)'5 C or
2'0
D
Figure 3, Effect of Sa~osin D on SDhin~omvelinase a n d G 1 u c o c e r e b r o s i d a s e , Various amounts of saposin D w e r e a d d e d to an assay mixture of s p h i n g o m y e l i n a s e and fl-glucosylceramidase activities and its effect was c o m p a r e d w i t h t h a t of saposin C. Sphingomyelinase activity assay mixture contained 20 nmol [methyl-14C] sphingomyelin (830 cpm/nmol), I0 lag Triton X-100, and 50 lamol acetate buffer pH 5.0 in 200 )II. Crude s p h i n g o m y e l i n a s e p r e p a r e d f r o m h u m a n p l a c e n t a (Sigma Chem. Co.) was used as enzyme source. Incubation was conducted at 37°C for I hr. 13-Glucosylceramidase a s s a y m i x t u r e c o n t a i n e d 5.25 n m o l to [ g l u c o - 3 H ) g l u c o c e r e b r o s i d e (1358 c p m / n m o l ) and 20 lamol c i t r a t e - p h o s p h a t e b u f f e r pH 4.2 in 200 lal. Human liver particulate preparation solubilized by 0.1% Triton X-100 was used as e n z y m e source. The activities for s p h i n g o m y e l i n a s e w i t h saposin D is s h o w n by open squares ( [ ] ) and t h a t w i t h saposin C is shown by closed squares ( • ) . The activity for 13-glucosylceramidase w i t h saposin D is e x p r e s s e d by o p e n circle ( O ) and t h a t w i t h s a p o s i n C is e x p r e s s e d by closed circle ( 11 ). No f u r t h e r i n c r e a s e of ~glucosylceramidase activity was observed by increasing saposins up to 35 lag each.
DISCUSSION In a c c o r d a n c e w i t h prosaposin generates i s o l a t i o n of a t h i r d
our recent
prediction that
the
proteolytic
4 s a p o s i n p r o t e i n s of s i m i l a r s t r u c t u r e ( 1 9 ) w e s a p o s i n p r o t e i n , s a p o s i n D, w h i c h
stimulates
processing
of
report here the the hydrolysis
of
s p h i n g o m y e l i n . P r o o f t h a t s a p o s i n D a r i s e s f r o m d o m a i n 4 of p r o s a p o s i n w a s o b t a i n e d by demonstrating and
the
addition,
c o l i n e a r i t y b e t w e e n its f i r s t 19 c h e m i c a l l y d e t e r m i n e d
deduced saposin
sequence D has
obtained
the
correct
from
domain
predicted
4 of t h e
molecular
a m i n o acids
prosaposin
weight
(10
cDNA. I n kDa)
p r o t e o l y t i c p r o c e s s i n g of d o m a i n 4. W h i l e t h i s w o r k w a s in p r o g r e s s F u r s t e t al. isolated the same protein from Gaucher spleen termed
component
from (31)
C; its a m i n o acid
s e q u e n c e is i d e n t i c a l t o s a p o s i n D e x c e p t f o r t h e a b s e n c e of t h e f i r s t t w o N - t e r m i n a l amino
acids which
may
have
been
removed
by
peptidase
a c t i o n . No a c t i v a t i n g
p r o p e r t i e s w e r e r e p o r t e d f o r c o m p o n e n t C. Saposin D has a remarkably
high sp e c i f i c i t y in s t i m u l a t i n g s p h i n g o m y e l i n a s e
a c t i v i t y . A c t i v a t i o n of s p h i n g o m y e l i n a s e a c t i v i t y b y s a p o s i n C h a s also b e e n r e p o r t e d 407
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I. Effect of Saposin D on GM 1 G a n g l i o s i d e g-Galactosidase, Galactocerebroside B-Galactosidase a n d 4 - M e t h y l u m b e l l i f e r y l Glucoside l~-G1ucosidase
Saposin
=,,
(~g)
nmol hydrolyzed/hr Saposin + Saposin
GMI G a n g l i o s i d e ~-galactosidase
B (42) D (45)
0 0
0.44 0.00
Galactosylceramidase
C (8) (16) (24)
0.41 0.41 0.41
0.57 1.24 1,48
D (14) (54) (95)
0.41 0.41 0.41
0.34 0.44 0.44
C (4) ( I 0) (25)
3.70 3.70 3.7O
9.00 t3.80 13.80
D (4) (10) (25)
3.70 3.70 3.70
5.50 5.00 5.00
4 - M e t h y l u m b e l l i f e r y l glucoside B - g l u c o s i d ase
(15); b u t in our h a n d s s a p o s i n C failed to s t i m u l a t e s p h i n g o m y e l i n a s e a c t i v i t y . Christomanou and her colleagues (20) purified two proteins, n a m e d A I and A 2, which stimulated
s p h i n g o m y e l i n a s e activity; t h e i r
A I p r o t e i n was f o u n d b y c h e m i c a l
s e q u e n c i n g to be i d e n t i c a l to saposin C (17). It is possible t h a t our HPLC-purified s a p o s i n C lost its s p h i n g o m y e l i n a s e a c t i v a t i n g p r o p e r t y
w h i l e r e t a i n i n g its tS-
g l u c o s y l c e r a m i d a s e s t i m u l a t i n g p r o p e r t y . A l t e r n a t i v e l y , p r e v i o u s p r e p a r a t i o n s of saposin C m a y have b e e n c o n t a m i n a t e d w i t h saposin D since their properties are v e r y s i m i l a r and e x t r e m e l y s m a l l a m o u n t s of s a p o s i n D are r e q u i r e d for a c t i v a t i o n . A n o t h e r e x p l a n a t i o n is the d e t e r g e n t - l i k e p r o p e r t y of s a p o s i n C w h i c h h e l p e d to solubilize s p h i n g o m y e l i n in the absence of Triton X-100. Prior to the discovery that saposin 13 and C are both d e r i v e d from the same p r e c u r s o r p r o t e i n (19), t h e s e proteins w e r e n a m e d n o n s y s t e m a t i c a l l y . The isolation of
saposin
D reported
nomenclature
for
the
here saposin
has
prompted
proteins.
As
us
to
propose
mentioned
the
a new
systematic
precursor
protein
( p r o s a p o s i n ) c o n t a i n s 4 d o m a i n s comprised of similar 80 a m i n o acid m o d u l a r units w h i c h are r e l e a s e d
u p o n proteolysis. We p r o p o s e t h a t t h o s e p r o t e i n s w h i c h are
derived from the first, second, third and f o u r t h domains be n a m e d saposin A, 13, C and D. Of the four, s a p o s i n A has y e t to be isolated. Partial proteolysis of prosaposin also 408
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
y i e l d s i n t e r m e d i a t e c l e a v a g e p r o d u c t s and t h e s e h a v e b e e n d e t e c t e d in most h u m a n t i s s u e s (19). We p r o p o s e t h a t p r o t e i n s p o s s e s s i n g two s a p o s i n d o m a i n s w i t h s u b u n i t molecular w e i g h t s of 2 5 - 3 0 kDa be called d i s a p o s i n A, B and C; containing d o m a i n s I and 2, 2 and 3, and 3 and 4 r e s p e c t i v e l y . S i m i l a r l y , c l e a v a g e p r o d u c t s c o n t a i n i n g t h r e e s a p o s i n d o m a i n s w i t h s u b u n i t m o l e c u l a r w e i g h t s of a p p r o x i m a t e l y 48 and 43 kDa, be n a m e d t r i s a p o s i n A and B; containing d o m a i n s 1,2 and 3 and d o m a i n s 2, 3, 4 r e s p e c t i v e l y . I n l i n e w i t h this proposal, w e h a v e r e c e n t l y i s o l a t e d a g l y c o p r o t e i n from Gaucher s p l e e n w h i c h a p p e a r s to be d i s a p o s i n A.
ACKNOWLEDGEMENTS We a r e g r a t e f u l to Dr. David W e n g e r for p r o v i d i n g us w i t h a n t i b o d i e s a g a i n s t saposin C and r a d i o a c t i v e sphingolipid s u b s t r a t e s , to Dr. A r y a n F l u h a r t y for giving us a n t i b o d i e s a g a i n s t s a p o s i n B,
to Dr. Yoshimi Y a m a m o t o for p u r i f i e d f}-galactosidase
and
GM I, and to Dr. N o r m a n R a d i n for s h o w i n g us an
radioactive
unpublished
ganglioside
manuscript.
We
also t h a n k
Susan
O'Brien for
her
assistance
in
p r e p a r a t i o n of this m a n u s c r i p t . This s t u d y w a s s u p p o r t e d in p a r t b y NIH r e s e a r c h grants NS-13559 (YK) and HD-18983 and NS-08682 (JOB).
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. I I. 12. 13. 14. 15. 16. 17. 18. 19.
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