(Received for publication, September 28, 1995; and in revised form, December 11, 1995)
From the
Saposin C is an essential co-factor for the hydrolysis of
glucosylceramide by acid -glucosidase in mammals. In addition,
prosaposin promotes neurite outgrowth in vitro via sequences
in saposin C. The regional organization of these neurotrophic and
activation properties of saposin C was elucidated using recombinant or
chemically synthesized saposin Cs from various regions of the molecule.
Unreduced and reduced proteins were analyzed by electrospray-mass
spectrometry to establish the complement of disulfide bonds in selected
saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins
containing various length segments of saposin B and C localized the
neurotrophic and acid
-glucosidase activation properties to the
carboxyl- and NH
-terminal 50% of saposin C, respectively.
The peptide spanning residues 22-31 had neurotrophic effects.
Molecular modeling and site-directed mutagenesis localized the
activation properties of saposin C to the region spanning residues
47-62. Secondary structure was needed for retention of this
property. Single substitutions of R and S at the conserved cysteines at
47 or 78 diminished but did not obliterate the activation properties.
These results indicate the segregation of neurotrophic and activation
properties of saposin C to two different faces of the molecule and
suggest a topographic sequestration of the activation region of
prosaposin for protection of the cell from adverse hydrolytic activity
of acid
-glucosidase.
The in vivo activities of several lysosomal hydrolases
require the presence of small (80 amino acids) heat stable
activator proteins or saposins(1, 2, 3) .
Saposin C was first described by Ho and O'Brien (4) in
1971 as a soluble factor that stimulated the activity of acid
-glucosidase (EC 3.2.1.45, N-acyl-sphingosyl-
-D-glucoside:glucohydrolase).
This saposin and three other highly homologous saposins, A, B and D,
are derived from the same precursor, prosaposin, by proteolytic
processing(5, 6, 7, 8) . Saposins A,
B, C, and D are encoded in the same reading frame of a primary
transcript (see Fig. 1A)(5, 6, 7) . In
addition to their highly similar sequences (
50% amino acid
identity), each saposin has six strictly conserved cysteines (see Fig. 1B). For each saposin the primary amino acid
sequences have been determined chemically and predicted from the
cDNA(5, 6, 7, 9) . Recently, the
disulfide structures of saposins B and C have been solved (10) , and those for saposins A and D are likely identical.
Figure 1: Schematic diagram (A) and sequences (B) of the prosaposin gene, cDNA, and amino acid residues. In A the genomic human DNA including the promoter (Y. Sun and G. A. Grabowski, unpublished observations) occupies about 35 kilobase pairs on chromosome 10q. The black rectangles represent the 14 exons, and the white horizontal bars are the intronic sequences. The initiating ATG is in the first exon. The saposin A, B, C, and D regions are indicated by similar shadings of regions from the gene and cDNA. The mouse genomic organization is essentially identical (Y. Sun and G. A. Grabowski, unpublished observations). In B is shown the saposin C amino acid sequence homology between several species. A derived consensus sequence for residues 47-63 is shown: h, hydrophobic amino acid; b, branch chain amino acid; a, aromatic amino acid; X, any amino acid. Placement of all cysteines and the N-glycosylation sites are conserved. The amino acid sequence of human saposin B is shown at the bottom.
Saposin B functions as a physiological solubilizer of the
glycosphingolipid substrates for arylsulfatase A (sulfatide, in
vivo), -galactosidase A (globotriaosylceramide, in
vitro), and
-galactosidase (G
-ganglioside, in vitro)(2) . The affinities of these substrates for
saposin B apparently forms the basis for its differential metabolic
effects, and a general model has been proposed for its mechanism of
action(2) . The physiologic functions of saposins A and D are
not defined, although in vitro and ex vivo studies
show that saposin D enhances ceramidase activity(11) .
Although saposin C is structurally similar to saposin B, the saposin
C mechanism of action is different and less completely defined. Saposin
C binds to and may transfer gangliosides between artificial membranes (12) , but it does not bind glucosylceramide, the acid
-glucosidase substrate(13) . Saposin C binds directly to
acid
-glucosidase (13) and may alter the structure of
negatively charged phospholipid, particularly phosphatidylserine,
interfaces(10) . These function to conform acid
-glucosidase and possibly alter binding at the lipid interface to
effect an enhancement of substrate hydrolytic
rates(13, 14) . Interaction of saposin C with acid
-glucosidase is complex and involves high (specific) and low
affinity sites(15) . The latter may interfere with a binding
site on the enzyme for phosphatidylserine, leading to diminished
activation(13, 15) . Recently, recombinant (15) and chemically synthesized (16) peptides were used
to regionally localize the activation and binding components of saposin
C to its carboxyl-terminal 50%.
During the conduct of these studies,
prosaposin was shown to have glycosphingolipid binding and transport
properties (12) and neurotrophic effects in
vitro(17) . The neurite outgrowth properties of prosaposin
appear to regionally localize to the NH-terminal 50% of
saposin C(18) . Consequently, the biological roles of
prosaposin may be multidimensional and include plasma membrane-mediated
signal transduction events as well as essential intracellular
glucosylceramide catabolic regulation. In this communication
recombinant and chemically synthesized saposin Cs are used to explore
the functional segregation of its neurotrophic and activation
properties. A variety of chimeric and mutagenized saposin Cs were
created to characterize residues critical to its neurotrophic effects
and its interaction with acid
-glucosidase.
Figure 2:
Schematic representation of the various
saposin C fragments. The mature saposin C NH-terminal amino
acid (see Fig. 1B) is designated 1. The nomenclature
used is as in the following example: saposin
C(1-41)B(42-80) means that the chimeric saposin contains
residues 1-41 from saposin C and residues 42-80 from
saposin B. MC refers to the mouse saposin C. The AC
value refers to the concentration of saposin required to achieve
50% of maximal acid
-glucosidase activation. The competition
column refers to the ability of saposins that do not activate acid
-glucosidase to decrease the activity of the enzyme in the
presence of 100 nM wild-type saposin C (15) . A minus
sign means that no effect was observed up to 500 nM. The
blanks indicate those saposin Cs that activated the enzyme and were not
tested as competitors. A depicts saposins that maintain the
complete sequence length of saposin C that were chimeric (rows
3-8) or point-mutated (rows 9-16). B shows fragments that were synthesized recombinantly (rows
1-5) or chemically (rows 6-8). Red. Sap.
C refers to the reduced and carboxymethylated saposin
C.
Expression of the saposins in E.
coli followed transformation into BL21(DE3) cells that contain a
isopropyl-1-thio--D-galactopyranoside inducible
T7-polymerase gene(15) . Transformations, incubations,
inductions with 1 mM isopropyl-1-thio-
-D-galactopyranoside, cell harvest,
and isolation of pure proteins were as described
previously(15) . Natural saposin C was purified from Gaucher
disease spleen as described(15) .
HPLC (C column) purification of the saposins was in 0.05% trifluoroacetic
acid with an acetonitrile gradient (0-40% in 5 min and then
40-75% in 35 min) and were monitored by UV detection at 225 nm.
The major peaks were collected and dried by rotatory evaporation and
subjected to NH
-terminal amino acid analysis by Edman
degradation and/or mass determination by electrospray-mass spectrometry
(ES-MS) (PE Sciex API-III, PE Sciex, Thornhill, Canada). A delivery
solvent of a methanol/water (1:1, v/v) with 0.1% formic acid was used
for flow injection experiments at a flow rate of 5 µl/min. Dried
proteins were reduced by dissolving with 6 M guanidine HCl in
50 mM Tris-HCl, pH 8.0. (0.2 ml) with dithiothreitol (1 M, 5 µl) for 3 h at 37 °C. The proteins were
reisolated by HPLC as above. The expected molecular weights were
calculated (DNAsis, Hitachi Software Engineering, San Bruno, CA) from
the predicted amino acid sequence of the fully reduced normal or mutant
saposin including the flanking vector derived NH
-terminal
amino acids and the carboxyl-terminal His-tag (see above).
Figure 3: Neurite outgrowth effects of saposin C derivatives on Neuro2A (A) and NS20Y (B) cells. 1 mM dibutyryl-cAMP (A) and 2.2 nM ciliary neurotrophic factor (B, CNTF) were used as controls for the positive effects in Neuro2A and NS20Y, respectively. The mean and standard errors are based on results from three separate experiments, each with over 200 cells counted. The absence of error bars means that the error was too small to display. The concentrations and saposin derivatives are shown below. n-Saposin C and r-saposin C refer to the natural and the recombinant saposin Cs, respectively.
In comparison, the acid -glucosidase activation effect
was mediated by the carboxyl-terminal 50% of saposin C ( Fig. 2and 4). The chimeric saposin B(1-41)C(42-80)
had
70% of the activation effect of wild-type saposin C. Saposin
C(1-41)B(42-80) was not active (Fig. 4A).
Saposin C(1-41), C(1-58) and saposin C(42-80) were
inactive (Fig. 2). These above results show that the
neurotrophic and acid
-glucosidase activation properties of
saposin C are segregate to the NH
- and carboxyl-terminal
50% of the molecule, respectively. Also, a linear peptide of residues
22-31 is sufficient for neurotrophic effects, but higher order
structures in the carboxyl-terminal 50% are needed for acid
-glucosidase activation effects.
Figure 4:
Activation profiles for acid
-glucosidase by chimeric (A), point-substituted (B), or cysteine-mutated (C) saposin Cs. All assays
were conducted in the presence of 2 nM acid
-glucosidase,
0.4 µg/ml phosphatidylserine, and 0.1 M sodium acetate, pH
4.7. Enzyme was preincubated with phosphatidylserine and saposin C
before substrate was added to start the reaction. The fold change
refers to the ratio of final to initial activity. The concentration of
the saposin was based on the concentration of the homogeneous protein
and the determined or calculated molecular weight. In A,
saposin C (
), saposin B(1-41)C(42-80) (&cjs2031;),
saposin C(1-41)MC(42-80) (
), and saposin
C(1-41)B(42-80) (
) are shown. In B, saposin
C (
), saposin C Q48W (*), Q48R (&cjs2031;), Q48N (
), Q48I
(
), and Q48A/E49A (
) are shown. In C, the typical
effects of cysteine mutated sapsosin Cs are shown. The results are
shown for wild-type saposin C (
), C72G (
), C47S (
),
C78R (
), and C78S (*).
Neither saposin B(1-52)C(53-80) nor
saposin C(1-52)B(53-80) enhanced acid -glucosidase
activity (Fig. 2A). Saposin C(1-52)B(53-80)
or C(1-41)B(42-80) up to 140 nM also did not
inhibit wild-type saposin C/acid
-glucosidase interaction (Fig. 2A). Saposin C(1-61)B(59-80)
activated acid
-glucosidase to the same level as the wild-type
saposin C, but the AC
was nearly 10-fold increased, i.e. residues 53-61 are important for the binding to but
not activation of acid
-glucosidase. Residues 47-50 are
strictly conserved in several species (Fig. 1B), but
the carboxyl-terminal 50% of the mouse and human saposin Cs differ at
19/39 residues. In the region of residues 48-61, 12 of 14 amino
acids are identical or highly similar. Using the cross-species saposin
C, HC(1-41)MC(42-80), the AC
was about
3-4-fold increased (
70 nMversus 15 nM wild-type) and nearly full activation (70-80%) could be
achieved. These results provide additional localization to amino acids
47-61 for essential residues for binding to and activation of
acid
-glucosidase by saposin C.
Chemically synthesized saposins
C(22-31), C(48-55), and C(53-63) did not interact
with acid -glucosidase (Fig. 2B). Saposin
C(22-31) was used as an internal control because the
NH
-terminal is not important for interaction with acid
-glucosidase. Other controls for nonspecific effects included
human angiotensin I, II, and III and bradykinin. Reduced and
carboxymethylated wild-type saposin C (15) (Fig. 2B) also did not interact with acid
-glucosidase. These findings showed that there is a requirement
for higher order structure of saposin C for interaction with acid
-glucosidase.
The importance of the highly conserved Gln and Glu
residues (Fig. 1B) was
evaluated by site-directed mutagenesis. Each of the expressed mutant
saposins that were available in sufficient amounts provided single
sharp peaks on HPLC and were in the fully oxidized forms (Table 1). The singly mutated (Q48N) or the doubly mutated
Q48A/E49A saposin Cs did not activate the enzyme nor did they compete
with wild-type saposin C (Fig. 2). The next series of point
substituted saposin Cs were created from the predicted secondary
structure (using the Robson algorithm) surrounding the
Gln
-Glu
sequence. As shown in Fig. 5A, wild-type saposin C is predicted to have a
major turn near Gln
-Glu
involving residues
52-55 (Fig. 5). By comparison, neither saposin B nor
saposin C Q48N or Q48A/E49A have this turn (Fig. 5B).
We assumed that this predicted turn was important for saposin C-acid
-glucosidase interaction. We modeled all possible substitutions at
Gln
and only the highly nonconservative substitutions,
Trp, Arg, and Ile were predicted to preserve the turn (Fig. 5C). Similar studies for amino acid substitutions
at residues 47-55 showed that Gln
was the least
tolerant of change for preservation of the turn; i.e. 10-15 different substitutions were tolerated at residues
other than Gln
. On this basis, saposin Cs Gln
Trp, Gln
Arg, and Gln
Ile were expressed and evaluated. At equimolar amounts
these saposins achieved about 70% of the wild-type saposin C
stimulation of the enzyme (Fig. 4B). The AC
values were 2.5-fold (Gln
Trp and Gln
Ile) to
4-fold (Gln
R) increased. These results were similar to those with
the saposins B(1-41)C(42-80) and
HC(1-41)MC(42-80), both of which retain the predicted turn.
These results indicate that the maintenance of the predicted turn is
critical to the functional interaction of saposin C and acid
-glucosidase but that other minor secondary structural changes
and/or the specific residues within the Gln
-Glu
region alter the affinity of saposin C for the enzyme and ability
to enhance acid
-glucosidase activity.
Figure 5:
Schematic of the predicted effects on
secondary structure for wild-type (A) or mutant (B and C) saposin Cs. The Robson algorithm was used. The
boundaries of a predicted helix, sheet, turn, or coil are indicated by
the amino acid residue number for ease of orientation. The amino acid
numbers refer to those from native saposin C without the additional
NH- and COOH-terminal sequences of the recombinant saposin
Cs. The first turns in A, B, and C include
these extra NH
-terminal amino acids. They have no long
range effects on predicted structures. A single major turn is predicted
to involve residues 52-55 of the wild-type sequence. In B this turn was eliminated by the substitution of Q48N and all other
amino acids, except Arg, Trp, and Ile (C). Other local
secondary structural changes are also predicted between the wild-type (A) and point-mutated (C) sequences. In the region of
the indicated turn, the calculations were not affected by the presence
or the absence of the nonsaposin sequences.
Singly substituted
saposin Cs with Arg or Ser at position 47 or 78 were used to examine
the role of disulfide bonds in the effects of saposin C on acid
-glucosidase. By ES-MS analyses each of these cysteine-mutated
saposin Cs had two disulfide bonds (Table 1). Although activation
(2.5-3-fold) of acid
-glucosidase was retained, this was
achieved only at concentrations greater than 150 nM (see Fig. 4C for typical results). These properties were
retained after heating these saposins at 95 °C for 10 min. With the
soluble forms of C72G or C72F saposin C, activation was essentially the
same as achieved with the wild-type saposin C (Fig. 4C). Previously, the C72F or C72G saposins were
used directly as Ni
column purified preparations, and
we observed inhibition of wild-type saposin Cs, effects. This was
reproducible but only with the preparations containing a large amount
of an aggregate. The nonaggregated forms of C72G or C72F activated acid
-glucosidase, by native polyacrylamide gel electrophoresis were
monomeric, and had the masses as shown in Table 1.
Saposin C was first isolated as a heat stable factor from the
spleens of Gaucher disease patients(4) . This 80-amino acid
peptide was thought to function simply as a co-factor for enhancing
acid -glucosidase cleavage of its substrates, glucosylceramide and
synthetic
-glucosides(15, 24, 25, 26, 27) ,
by binding to the enzyme(13, 28) . This physiologic
role was confirmed by the presence of a Gaucher disease-like phenotype
and glucosylceramide storage in patients with deficient saposin
C(29, 30, 31, 32) .
Phosphatidylserine modified the effect of saposin C because low or high
concentrations increased or diminished enhancement of activity,
respectively(13, 15) . Qi et al.(15) showed that there is highly specific (nanomolar)
interaction of saposin C with acid
-glucosidase and showed the
importance of a folded structure and the carboxyl-terminal 50% for this
specific effect. Recently, saposin C was shown to have neurotrophic
effects when placed in the medium of neuroblastoma cell
lines(17, 18) . The current investigations showed the
segregation of the different functions of saposin C to discrete regions
of the protein.
Previously, we showed that recombinant saposin Cs
produced in E. coli had properties nearly identical to those
of the natural wild-type protein(15) . Importantly, for the
present studies, HPLC and ES-MS analyses provided insight into the
structure of the expressed saposins from E. coli. This was
important because we had assumed that the conservation of cysteine
placement in human and mouse saposins B and C would preserve the
formation of disulfide bonds in chimeric saposins composed of
components of B and C. In all of the mutant saposins containing the
complete sequence and not mutagenized at the cysteines, small yields of
saposins with aberrant disulfide structure were detected by HPLC and
ES-MS. The isolation and use of fully oxidized saposin C allowed direct
comparisons of the properties of wild-type and mutant proteins.
Introduction of mutations at cysteines 47 and 78 or cysteine 72 led to
a large proportion of the mutant saposins being expressed in inclusion
bodies or as aggregates, respectively. This required direct HPLC
purification from TFA-solubilized pellets of the Cys and
Cys
mutants. These soluble mutant saposins retained two
disulfide bonds. Similarly, monomeric C47G or C47F had normal
activation properties, but these proteins aggregated easily. The
cysteine-substituted (Arg or Ser) saposin Cs were heated at 95 °C
and retained their abnormal acid
-glucosidase activating
properties, even though one disulfide bond was disrupted. These results
suggest that not all of the disulfide bonds are necessary for the
retention of thermal stability properties as assessed in this manner.
Our previous work showed that reduction and carboxymethylation
destroyed the activating and binding properties of wild-type saposin
C(15) . This result was confirmed(16) . Taken together,
these results indicate the need for some disulfide structure for
retention of these properties. The fact that the Arg- (nonconservative)
or Ser-substituted (conservative and isosteric) saposin Cs at position
47 or 78 had lost their high affinity interaction with acid
-glucosidase indicates the importance of the disulfide bonds
rather than the nature amino acid at these residues alone.
The
activation effects of saposin C on acid -glucosidase were
regionally localized to the carboxyl-terminal 50% using chimeric and
point-mutated proteins. The inactivity of saposin
C(1-52)B(53-80) and the activity of saposin
C(1-61)B(59-80) as well as the high conservation of
residues 47-53 (Fig. 1B) indicated the importance
of the region spanning amino acids 47-61. Saposin C (Q48N) had an
isofunctional substitution but was inactive. Saposin Cs Q48W, Q48R, and
Q48I retain activity even though these substitutions are highly
nonconservative and disruptive(33) . However, only Trp, Ile,
and Arg substitutions were predicted to preserve the turn involving
residues 52-55. These mutants did retain the activation and
binding properties of saposin C but with decreased apparent affinity
for the enzyme. The similarity of the results obtained with of the
human/mouse saposin C/C and human/human chimeric saposin B/Cs showed
that the composition of the amino acids within the region of
43-59 and the structure assumed by the folded molecule were
equally important to retention of this property. Apparently, this
structure is, at least in part, dictated by a consensus sequence
spanning amino acids 47-63 (Fig. 1B). This
consensus was derived from the four saposin Cs that have been shown to
activate human acid
-glucosidase(9, 34, 35, 36) . (
)
The conservation of cysteine placement in the saposins
has suggested that the disulfide structure of saposin C is essential
for the activation of acid -glucosidase by saposin C(10) .
Our present results suggest otherwise. The major effect of Arg or Ser
substitution at cysteine 47 or 78 was to greatly increase the AC
values with minor effects on the maximal levels of stimulation.
This indicates the need for at most two disulfide bonds in saposin C
for retention of activity. Furthermore, secondary structure in the
carboxyl-terminal 50% of saposin C is important but apparently not
sufficient for specific interaction with acid
-glucosidase.
Recently, Weiler et al.(16) used chemically
synthesized saposin C peptides to suggest that the saposin C activation
effects toward acid -glucosidase are spread throughout the entire
molecule. Those results implied that the disulfide structure was not
necessary for the activation properties of saposin C. Weiler et al.(16) obtained AC
values 3-6 orders of
magnitude greater than ours and those extrapolated from the data of
others(13, 15) . We could achieve only low levels of
saposin C effects at µM amounts using their assay system.
In addition, the lack of nonsaposin C peptide controls in the studies
of Weiler et al.(16) makes it difficult to exclude
nonspecific interactions between their synthetic peptides and the
hydrophobic acid
-glucosidase. Our findings of: 1) highly
selective regional localization of activation effects to the
carboxyl-terminal 50% of saposin C, 2) the clear lack of effect of the
NH
-terminal 50%, and 3) the inability of various saposin C
and nonsaposin C peptides to activate acid
-glucosidase indicate
that a much more specific interaction was examined in the present
studies.
The localization of the neurotrophic effects to the
NH-terminal 50% of saposin C is consistent with the recent
results of O'Brien et al.(17, 18) .
Indeed, the neurotrophic effect of prosaposin was sublocalized to the
saposin C residues 13-29, and 18-29 were suggested as
essential. Our studies are consistent with that suggestion because we
found that residues 22-31 retain neurotrophic properties. This
result refines the localization to residues 22-29. Indeed, on an
equimolar basis, our saposin C(22-31) provided similar degrees of
neurite outgrowth as did the comparable saposins C(8-29) or
C(18-34) of O'Brien et al.(18) . These
results indicate that a linear 7-11 residue sequence is
sufficient for the neurotrophic effects of saposin C and prosaposin but
that longer sequences from the NH
-terminal half of saposin
C enhance the interaction with NS20Y cells; i.e. probably
increase the affinity for the receptor. The identical effects of the
natural (glycosylated) and recombinant (unglycosylated) saposin Cs show
that the enhanced effect of longer sequences is not due to the
oligosaccharide moiety. Importantly, the neurite outgrowth effects are
cell-specific because NS20Y, a cholinergic neuroblastoma cell line,
responded better than Neuro2A, a noncholinergic cell line(23) .
This is consistent with the localization of prosaposin, as opposed to
the individual saposins, to cholinergic neurons in rat
brain(37) . These results imply that the prosaposin-mediated
neurotrophic effects, via its saposin C segment, may be mainly in
specific sets of neurons.
The segregation of neurotrophic and
activation regions of saposin C to the NH-terminal and
carboxyl-terminal 50% regions, respectively, expands the repertoire of
prosaposin functions and its functional organization. The schematic
presented in Fig. 6suggests that these different functions
occupy opposite faces of the saposin C molecule. Prosaposin does not
substantially activate acid
-glucosidase(15) , but it does
have neurotrophic effects(17) . (
)These results
suggest that occupancy of the N-glycosylation site of saposin
C may keep its NH
-terminal region exposed and that the
carboxyl-terminal region is hydrophobic and buried in prosaposin. This
could provide an interesting topographic mechanism for controlling acid
-glucosidase activity during transit through nonlysosomal
compartments, i.e. acid
-glucosidase would be active only
in the lysosome where saposin C is proteolytically liberated from
prosaposin(38, 39) . In addition, the temporal and
spatial regulation of prosaposin expression throughout development (40) and its tissue specific processing indicate that this
multifunctional locus encodes biologic properties that extend beyond
its initial role as a lysosomal hydrolase activator precursor.
Figure 6:
A schematic of the functional
organization of neurotrophic and acid -glucosidase activation
properties of saposin C. Except for the box that indicates the
predicted turn in Fig. 5A and the disulfide bonds, the
figure is not meant to represent known physical structure. The darkly highlighted residues from 13-33 encompass the
neurotrophic region. The heavily circled residues,
22-32, are of major significance to this effect. The minimal
peptide may be as small as residues, 22-29. The region spanning
residues 47-63 is important to the acid
-glucosidase
activation effects of saposin C, and the presence of all three
disulfide bonds is also important for this interaction. Higher order
structure is also required to have full activation effects on acid
-glucosidase.