(Received for publication, October 2, 1995; and in revised form, February 22, 1996)
From the
Distinct from the noncovalently linked recombinant human stem
cell factor (rhSCF) dimer, we report here the isolation and
identification of an SDS-nondissociable dimer produced during
folding/oxidation of rhSCF. Experimental evidence using various
cleavage strategies and analyses shows that the isolated dimer is
composed of two rhSCF monomers covalently linked by four disulfide
bonds. The cysteines are paired as in the noncovalently associated
dimer except that all pairings are intermolecular rather than
intramolecular. Other structural models, involving intertwining of
intramolecular disulfide loops, are ruled out. The molecule behaves
similarly to the noncovalently associated dimer during ion-exchange or
gel permeation chromatography. However, the disulfide-linked dimer
exhibits increased hydrophobicity in reverse-phase columns and in the
native state does not undergo spontaneous dimer
dissociation-association as seen for the noncovalent dimer.
Spectroscopic analyses indicate that the disulfide-linked and
noncovalently associated rhSCF dimers have grossly similar secondary
and tertiary structures. In vitro, the disulfide-linked dimer
exhibits approximately 3-fold higher biological activity in supporting
growth of a hematopoietic cell line and stimulating hematopoietic cell
colony formation from enriched human CD34 cells. The
molecule binds to the rhSCF receptor, Kit, with an efficiency only half
that of the noncovalently associated dimer. Formation of intermolecular
disulfides in the disulfide-linked dimer with retention of biological
activity has implications for the three-dimensional structure of
noncovalently held dimer and disulfide-linked dimer.
Stem cell factor (SCF), ()also termed ``kit
ligand'' or ``mast cell growth factor''(1, 2, 3, 4, 5, 6) functions
in the early stages of hematopoiesis and is also involved in the
development and function of other cell lineages, including melanocytes
and germ cells(7, 8) . SCF is initially synthesized as
membrane-bound forms of 248 or 220 amino acids, depending on
alternative splicing of exon 6. A soluble SCF form of 165 amino acids
is biologically functional, apparently arising by proteolytic release
from the extracellular domain of the membrane-bound 248-amino acid
SCF(9, 10, 11) . The naturally occurring
soluble SCF is glycosylated at both N-linked and O-linked sites(12, 13) .
SCF binds to its
receptor, Kit, to elicit its specific biological
functions(1, 2, 3) . The Kit receptor belongs
to the type III tyrosine kinase family whose members include receptors
for M-CSF and PDGF (14, 15, 16) . SCF, M-CSF,
and PDGF are all dimeric ligands (17, 18, 19, 20) that mediate
receptor
dimerization(17, 18, 19, 20) . We
have previously described the isolation and characterization of soluble
SCF recombinantly expressed in Escherichia
coli in a nonglycosylated form (rhSCF), and by CHO cells in a
glycosylated form (11, 17, 18) . These SCFs
are fully native and biologically functional. In contrast with the
M-CSF and PDGF dimers whose monomers are
disulfide-linked(19, 20, 21) , both
glycosylated and nonglycosylated SCF dimers contain noncovalently
linked monomers(12, 17) . The SCF noncovalently
associated dimer was observed to undergo spontaneous
dissociation-reassociation of monomers in its native
state(22) .
In a companion paper(23) , we described the isolation and characterization of intermediates derived during folding and oxidation of the reduced and denatured rhSCF and the assignment of a predominant in vitro folding pathway. The major folded SCF is the noncovalently linked dimer (SDS-dissociable) and a small fraction is SDS-nondissociable dimer. In the present study, we isolate the nondissociable dimer to apparent purity and demonstrate that it is biologically functional and is covalently linked by four intermolecular disulfide bonds involving all cysteinyl residues. The biological, biochemical, biophysical, and structural properties of the noncovalently and covalently linked dimers are compared, and the results provide some insights to the structure and function of SCF.
A
complete CNBr cleavage at the Met residues of SCF dimer species or
HO
-oxidized SCF dimer species was performed as
follows. Vacuum-dried samples were redissolved in 70% formic acid (0.2
mg in 150 µl) and then incubated with freshly prepared CNBr (400
molar ratio to SCF) at 25 °C for 24 h in the dark. For partial CNBr
cleavages, 50-fold molar excess of CNBr was used and the incubation
times were shortened to 2-8 h. All the cleaved samples were
immediately vacuum dried for further analysis.
Aliquots of dried samples (5-20 µg) were loaded onto individual lanes of precast 16% Laemmli polyacrylamide gels (10 wells; Novex Inc., San Diego, CA) and electrophoresed (25) under nonreducing and reducing conditions. After Coomassie Blue staining and destaining, protein band intensity in each gel lane was measured using an image scanner (PDI Inc., New York); images were integrated using PDQuest software (PDI Inc.). In separate analyses, gel bands were also electrophoretically transferred onto PVDF membrane and the Coomassie Blue-stained bands were excised for N-terminal sequence analysis (24) .
Figure 1: SDS-PAGE analyses and chromatographic separation of SDS-dissociable and nondissociable dimers of rhSCF. A, SDS-PAGE analyses. Lanes 1-3 (nonreducing), two SCF fractions after S Sepharose chromatography (30 µg each); and purified SDS-dissociable rhSCF, respectively; lanes 4 and 5 (reducing), purified SDS-dissociable and nondissociable rhSCF dimers, respectively (10 µg each); lanes 6 and 7, as lanes 4 and 5 (respectively), but nonreducing. Molecular mass markers are shown to the left of lane 1 (67, 45, 30, 21, and 14.5 kDa, from the top) and lane 4 (90, 67, 45, 30, 21, and 14.5 kDa, from the top). The arrowheads indicate the migration positions (nonreducing) of SDS-nondissociable dimer (upper arrows) and SDS-dissociable dimer (lower arrows). B, C4 reverse-phase chromatography of rhSCF pooled after S Sepharose chromatography. See ``Experimental Procedures'' for details. The thinner line represents percent B. C, analytical C4 reverse-phase HPLC. From top to bottom, the applied samples (25 µg each) were: rhSCF pooled after S Sepharose chromatography; pooled peak 1 material (SDS-dissociable dimer) after C4 reverse-phase chromatography as in panel B; pooled peak 2 material (SDS-nondissociable dimer) after C4 reverse-phase chromatography as in panel B. The absorption full scale is 0.5 at 215 nm.
Preparative reverse-phase column chromatography resolved these two species (Fig. 1B). Peak 1 eluting earlier represents the rhSCF which is 18.5 kDa on nonreducing SDS-PAGE and on reducing SDS-PAGE (Fig. 1A, lanes 4 and 6; note that the reduced protein migrates slightly faster than the nonreduced, as described previously (17) and as is typical for proteins containing intramolecular disulfide bonds). This form corresponds to the active noncovalently associated, SDS-dissociable rhSCF dimer(17) . Peak 2 represents the SCF dimer which is 37 kDa on nonreducing SDS-PAGE and about 18.5 kDa upon reducing SDS-PAGE (Fig. 1A, lanes 7 and 5, respectively). Analytical reverse-phase HPLC using a TFA-acetonitrile gradient elution is shown in Fig. 1C (bottom chromatogram). This analysis provides a full resolution of the two species and accurately estimates that the disulfide-linked dimer form is 10-20% of the total (varying somewhat between preparations) (top chromatogram). The middle and bottom chromatograms demonstrate the purity of both peaks obtained from the preparative C4 chromatography.
Figure 2: Endoproteinase Asp N-derived peptide maps of SDS-dissociable rhSCF dimer (A) and SDS-nondissociable rhSCF dimer (B). In each case 50 µg of digested material was analyzed. The elution positions of the two disulfide-containing peptides are indicated.
Figure 3: Possible structures for SDS-nondissociable rhSCF dimer. A, disulfide-linked dimers (A1, A2, and A3); the numbers -1, 26, 37, 48, and 159 shown in A1 are Met residues, and 4, 43, 89, and 138 are Cys residues. B, concatenated dimers (B1, B2, B3, B4, and B5).
These two types of models are intermolecularly disulfide-linked dimers and concatenated dimers (Fig. 3, A and B, respectively). There are three disulfide-linked dimers, of which cysteines are involved in the formation of four intermolecular S-S linkages (structure A1), or two inter- and two intramolecular S-S bridges (structures A2 and A3). The five possible concatenated dimers would contain interlocked, but not covalently linked, monomers (Fig. 3B). Structure B1 is a dimer concatenated by N-terminal disulfide loops of the two monomers, while structure B2 is interlocked by two C-terminal disulfide loops. Structures B3 and B4 are dimers with a respective N- and C-terminal disulfide loop of one monomer locked into the other monomer near a sequence region (between residues 44 and 88) shared by both N- and C-terminal disulfide loops. Structure B5 is concatenated between the N-terminal disulfide loop of one monomer and the C-terminal loop of the other. In order to determine which structure corresponds to the isolated SDS-nondissociable dimer, the experiments described in the following five sections were performed.
Figure 4: SDS-PAGE analyses of peptide products of SDS-nondissociable rhSCF dimer derived from chemical and proteolytic cleavages. Unless stated otherwise, all single cleavage products were run at 20-µg load and untreated SCF at 5-µg load. A, complete CNBr cleavage (24 h). Lane 1 (nonreducing), untreated; lanes 2 and 3 (nonreducing and reducing, respectively), cleavage products. B, partial CNBr cleavage (2 h). Lane 1 (nonreducing), untreated; lanes 2 and 3 (nonreducing and reducing, respectively), cleavage products. C, partial CNBr cleavage (8 h). Lane 2 (nonreducing) untreated; lanes 1 and 3 (nonreducing and reducing, respectively), cleavage products. D, endoproteinase Lys-C digestion. Lanes 1 and 2 (nonreducing), products at loads of 40 and 10 µg; lanes 3 and 4, as lanes 1 and 2, but reducing. E, BNPS-skatole cleavage. Lanes 1 and 2 (nonreducing), cleavage products at loads of 40 and 20 µg. F, complete CNBr cleavage of Met-oxidized dimer. Lane 1 (nonreducing), untreated Met-oxidized dimer; lane 2 (nonreducing), cleavage product; lanes 3 and 4, as lanes 1 and 2, but reducing.
In separate experiments, limited CNBr cleavages of the SDS
nondissociable rhSCF dimer were performed to help distinguish between
the structural models. It can be seen in Fig. 4(B and C) and in Table 1, that the incomplete cleavages lead to
bands about 17 kDa and about 35 kDa on nonreducing SDS-PAGE; the
relative amounts of these bands depend on the degree of partial
cleavage. From the sequence data of Table 1, it appears that the
17-kDa bands represent material cleaved at all Met residues. The 35-kDa
bands represent material cleaved at Met residues -1, 27, and 36
(note that the Ile to Met
peptide is lost in
the gel analysis and therefore not detected). The key point is that
this 35-kDa material is not cleaved at Met
since the
sequence for the peptide beginning at Val
is low or
missing. Therefore, the disulfide loop formed by
Cys
-Cys
disulfide bonding would be
opened up, but that formed by the Cys
-Cys
disulfide bond would not. The fact that this material retains the
size of 35 kDa on nonreducing SDS-PAGE is inconsistent with structures
B1, B3, and B5, but consistent with all the other structures, as
indicated in Table 2.
Figure 5:
A, partial DTT reduction of
SDS-dissociable rhSCF dimer (top panel) and SDS-nondissociable
rhSCF dimer (bottom panel). N and dimer have no
disulfides reduced; I-1 and I-2 are forms with one
disulfide reduced (see (23) ); a and b are
partially reduced, with the Cys-Cys
bond still intact; R refers to completely reduced
material. B, nonreducing SDS-PAGE of forms referred to in A. Lanes, from left to right, molecular mass markers (90, 67,
45, 30, 21, and 14.5 kDa, from the top), R, N, dimer, I-2, a,
b.
As summarized in Table 2, the only structure for the SDS-nondissociable rhSCF dimer compatible with all the results of the last five sections is structure A1, with four intermolecular disulfide bonds involving all four Cys residues of each monomer. Therefore, we will subsequently refer to the SDS-nondissociable dimer as disulfide-linked dimer.
The noncovalently associated rhSCF dimer and the disulfide-linked dimer have identical elution times and elution profiles on high resolution ion-exchange HPLC and gel filtration, indicating that they have similar surface charge distribution and molecular size in solution.
Figure 6: Spectroscopic properties of disulfide-linked rhSCF dimer (thin lines) and noncovalently associated rhSCF dimer (heavy lines). A, far UV CD spectra; B, near UV CD spectra; C, thermostability monitored by CD (ellipticity at 222 nm); and D, fluorescence spectra.
Figure 7: Biological properties of disulfide-linked rhSCF dimer (circles) and noncovalently associated rhSCF dimer (squares). A, stimulation of UT-7 cell proliferation; B, stimulation of GM-CFC colony formation; C, stimulation of BFU-E colony formation; and D, Kit receptor binding.
It
has been repeatedly shown previously that SCF acts synergistically with
later-acting hematopoietic cytokines to stimulate in vitro colony formation from hematopoietic progenitor
cells(2, 26) . For example, with purified
CD34 bone marrow cells, BFU-E colony formation is
minimal with SCF or EPO alone, but is marked with SCF and EPO together;
similarly GM-CFC colony formation is minimal with SCF or rhG-CSF alone,
but marked with SCF and rhG-CSF together. Dose-response curves for the
two rhSCF dimer species are shown in Fig. 7B (BFU-E;
fixed concentration of EPO) and Fig. 7C (GM-CFC; fixed
concentration of G-CSF). In both assays, the disulfide-linked dimer has
a potency severalfold greater than that of noncovalently associated SCF
dimer.
In contrast to the situation with biological potencies on
cells, the receptor binding affinity of disulfide-linked rhSCF is about
2-fold lower than that of noncovalently associated rhSCF. This is
demonstrated in Fig. 7D, in which the abilities of the
rhSCF species to compete with I-labeled rhSCF for binding
to receptor are determined.
Biologically active rhSCF can be recovered after folding and oxidation of inactive rhSCF in the solubilized extracts prepared from inclusion bodies after recombinant expression in bacteria(17, 18, 23) . The major folded/oxidized form is the SDS-dissociable, noncovalently associated dimer, corresponding to naturally occurring SCF. However, a significant amount of the rhSCF is the SDS nondissociable dimer that is separable from the main rhSCF form by reverse-phase chromatography. Our data allow us to propose and distinguish between eight different structural models that could explain why the minor form gives peptide mapping results (including disulfide-linked peptides) the same as those of the major form, but is SDS-nondissociable. As we show, structure A1, with the four intermolecular disulfide bonds, appears to represent the actual material.
As described in our companion study(23) ,
intermediate I-1 with a Cys-Cys
disulfide bond is the main intermediate form during rhSCF folding
and oxidation. This and other intermediates lead to the noncovalently
associated SCF dimer with intramolecular disulfides, but could also
undergo disulfide rearrangement to form intermolecular disulfides. For
such events to occur, the partially oxidized rhSCF monomers would have
to be associated prior to disulfide formation; we have shown that all
of the intermediate forms that have been identified are in dimeric
state (23) .
Many of the biochemical and biophysical
properties of the noncovalently associated dimer and the
disulfide-linked dimer appear indistinguishable, including surface
charge, molecular size, plus secondary and tertiary structure and local
environments. The disulfide-linked dimer does behave differently than
the noncovalently associated dimer on reverse-phase HPLC at low pH and
in the monomer dissociation-reassociation experiments. In each case the
differences essentially reflect the covalent attachment of the
disulfide-linked dimer. For example, with the reverse-phase HPLC, the
noncovalently associated SCF dissociates to monomer at the low pH, ()but the disulfide-linked SCF dimer is obviously unable to
dissociate.
The biological properties of the covalent dimer are
noteworthy. Its activity toward hematopoietic target cells is 3-fold
higher than the activity of noncovalently associated dimer. However, in
Kit receptor binding experiments, the disulfide-linked dimer if
anything displayed slightly lower affinity for Kit in comparison with
the noncovalently associated dimer. How can similar (or lower) receptor
binding be reconciled with higher biological activities for the
disulfide-linked dimer? Dose-response for SCF in the in vitro biological assays occurs in the 0.05-2 ng/ml concentration
range. In the case of the noncovalently associated dimer, depending on
the K for monomer association to dimer, it is
possible that much of the SCF could be monomeric at the 0.05-2
ng/ml concentration range. Lev et al. (29) have
proposed that monomeric SCF can mediate the dimerization and activation
of Kit receptor, but it remains possible that SCF dimer is necessary to
mediate Kit dimerization, or at least that SCF dimer may be more
effective at doing so than SCF monomer. Since the disulfide-linked SCF
dimer would of course be dimeric at all concentrations, it follows that
it could be more potent, i.e. more active at low
concentrations, than the dissociable dimer.
This line of reasoning
implies that the overall quaternary structure, including interactions
at the dimer interface, would be similar for the disulfide-linked and
noncovalently associated dimers. In considering the structure of SCF,
there are many reasons to expect similarity to the structure of M-CSF,
which is known(30) . As mentioned in the Introduction, SCF and
M-CSF are noncovalently associated and disulfide-linked dimers,
respectively, and their receptors are related members of the type III
tyrosine kinase family. As Bazan (31) has pointed out, the
exons of the SCF and M-CSF genes can be closely aligned, suggesting
evolutionary relatedness. Both SCF and M-CSF are expressed as longer
membrane-bound forms from which soluble forms are released by
proteolytic cleavage at sites encoded within exon 6, or as shorter
membrane-bound forms (lacking the exon 6-encoded cleavage sites as a
result of alternative mRNA splicing) which remain
membrane-associated(9, 10, 11, 32) .
The M-CSF monomer has intramolecular disulfide bonds
(Cys-Cys
and
Cys
-Cys
) (21, 30) that align well with those of the SCF monomer
(Cys
-Cys
and
Cys
-Cys
). M-CSF has a third
intramolecular disulfide (Cys
-Cys
) (21, 30) and an intermolecular disulfide joining the
Cys
residues of each monomer in the M-CSF dimer. These
disulfide bonds are apparent in the x-ray crystallographic structure of
M-CSF dimer(30) . The structure includes the four-helix bundle
for each monomer which had been proposed by Bazan (31) for both
M-CSF and SCF. The two monomers of M-CSF associate in head-to-head
fashion, i.e. the top ends of the helix bundles associate
leading to a flat and elongated overall shape. The SCF-equivalent
intramolecular disulfide bonds (Cys
-Cys
and Cys
-Cys
) of M-CSF are at the
ends of the helix bundles distal to the dimer interface, and the
Cys
-Cys
disulfide bond is of course
part of the interface.
Given that the disulfide-linked SCF dimer described here is highly active, we suggest the following speculation as to how its structure may compare to that of the noncovalently associated SCF dimer. If the quaternary structure of noncovalently associated SCF is homologous to that of M-CSF, the monomers of the disulfide-linked dimer would need to be inverted in order to accommodate the disulfide bond formation, without an adverse effect on activity. Alternatively, if the quaternary structures of the disulfide-linked SCF dimer and the noncovalently associated SCF dimer were similar to each other, both could be inverted relative to that of the M-CSF dimer. Third, and perhaps most likely, the quaternary structures of the SCFs could be similar to each other and to that of M-CSF if, for the disulfide-linked SCF dimer, the proposed A and D helices (31) were swapped between the monomers within the dimer. Such swapping would be feasible within the constraints of the proposed SCF structure (i.e. similar to M-CSF structure), could conceivably arise during the refolding of the E. coli-derived recombinant molecule, and would allow the observed intermolecular disulfide bond formation. There is precedent for such swapping of helices or other domains between monomers within overall oligomeric structures, e.g. interleukin 5 (33) and many other proteins as well(34) .