(Received for publication, April 2, 1997)
From Amgen, Inc., Thousand Oaks, California 91320-1789
We readily produced recombinant pro-macrophage
stimulating protein in a mammalian expression system, but it was only
weakly active after proteolytic activation. Active macrophage
stimulating protein is a disulfide-bonded heterodimer, but in our
hands, the subunits of recombinant macrophage stimulating protein were
mostly not disulfide bonded. Molecular modeling of the serine
proteinase domain of macrophage stimulating protein based on homology
to human trypsin suggested that macrophage stimulating protein, but not
plasminogen or hepatocyte growth factor, has a Cys residue (672) in
close proximity to the Cys residue (578) that forms the intersubunit
disulfide link with the other subunit. We hypothesized that
Cys672 might interfere with intersubunit disulfide
formation by forming an intrasubunit disulfide with
Cys578 and therefore mutated Cys672 to Ala.
After kallikrein activation, the subunits of Cys672 Ala
macrophage stimulating protein were fully disulfide linked, and the
mutant macrophage stimulating protein had 10-20-fold higher specific
activity than the wild type recombinant macrophage stimulating protein.
Macrophage-stimulating protein (MSP)1
was originally purified from human serum as a protein that enhances the
chemotactic response of murine peritoneal macrophages to the C5a
fraction of complement (1). In a screen for proteins that contain
kringle domains, Degen and co-workers (2) identified a cDNA that
was most closely related to hepatocyte growth factor (HGF) and hence
named it HGF-like (HGF-L). MSP and HGF-L were later shown to be the
same molecule (3). MSP and HGF (4) are plasminogen-related growth
factors and appear to exert their biological effects as ligands for the receptor protein tyrosine kinases RON (5, 6) and MET (7), respectively.
The murine orthologue of human RON is also known as STK (8, 9). Like
plasminogen, these growth factors require proteolytic activation for
biological activity, which occurs between homologous Arg-Val residues
and generates heterodimeric disulfide-bonded subunits (10). The subunit contains four kringle domains, whereas the
subunit contains
an inactive serine protease domain (SPD). It is not established which
protease is responsible for the in vivo activation of MSP,
but several proteases, such as human plasma kallikrein, are reported to
efficiently activate MSP in vitro (10). The biological role
of the RON/MSP cognate receptor/ligand pair is still unclear.
Experimental strategies to address this issue include analysis of the
expression pattern of RON in
situ,2 and a survey of the effects of
MSP in a variety of in vitro bioassays. Because the latter
requires a steady supply of MSP, we began producing recombinant MSP.
Our initial attempts to produce recombinant MSP generated preparations
with a dramatically reduced specific activity (~50-fold) as compared
with material that was purified from a nonrecombinant source. The
following studies were undertaken in attempts to determine the
underlying mechanism for the reduced activity and to investigate how
the activity might be restored to levels observed with naturally
produced protein.
The human MSP cDNA
(GenBankTM accession number L11924[GenBank] (11)) was a gift of Professor
Sandra Degen, University of Cincinnati. The Cys672 Ala
MSP mutant was constructed by polymerase chain reaction from the MSP
cDNA with a mutant primer containing a change from TG to GC at
nucleotides 2024 and 2025 (5
-CACAACGCCTGGGTCCTGGAAG-3
) and
a downstream primer (5
-CTGGCAACTAGAAGGCACAGTCG-3
) complementary to
vector pCDNA3 (Invitrogen, San Diego, CA). All contructs were verified by DNA sequencing.
Chinese hamster ovary (CHO) cells
deficient in dihydrofolate reductase activity (CHOd; Ref.
12) were transfected with calcium phosphate using pDSR
2-derived expression vectors (13) containing the wild type human MSP or mutant
human MSP cDNAs. Transfected colonies were cloned and analyzed by
Western blot using an antibody directed against Escherichia coli derived human MSP. High expressing clones for wild type MSP and mutant MSP were chosen for further analysis. CHOd
cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, nonessential amino acids, glutamine, penicillin and streptomycin, and hypoxanthine/thymidine. Transfected clones were grown in similar medium using 5% dialyzed fetal bovine serum and lacking hypoxanthine/thymidine. Large scale generation of conditioned medium was accomplished by seeding
transfected CHOd
clones into roller bottles containing
50% DMEM, 50% Ham's F-12 medium supplemented with 5% dialyzed fetal
bovine serum, nonessential amino acids, and glutamine. At 70%
confluence, cells were rinsed with phosphate-buffered saline and were
used to condition medium, as above, but lacking serum, for 5 days.
Conditioned medium, with or without concentration by diafiltration and without salt or pH adjustment, was chromatographed by absorption onto heparin-Sepharose (Pharmacia Biotech, Inc.) and eluted with a salt gradient in 20 mM sodium phosphate, pH 7. MSP-containing fractions were identified by Western blotting with rabbit anti-murine proMSP serum. MSP eluted at 0.4 M NaCl. Pooled fractions were dialyzed with 0.02 M Tris, pH 8.5, chromatographed by absorption on Q Sepharose HP (Pharmacia Biotech, Inc.), and eluted with a salt gradient in 0.02 M Tris, pH 8.5. MSP eluted at 0.1 M salt. MSP was activated either by incubation at 37 °C for 1 h with 10 µg/ml human kallikrein (Enzyme Research Laboratories, Inc., South Bend, IN), followed by addition of 1 mM Pefabloc (Boehringer Mannheim), or by passing MSP through a column of kallikrein coupled to cyanogen bromide activated-Sepharose (Pharmacia Biotech, Inc.) at a concentration of 1 mg/ml. Activated samples were dialyzed versus phosphate-buffered saline. N-terminal sequencing was performed on an Immobilon-PSQ (Millipore, Bedford, MA) blot using a Procise 494 sequencer (PE-ABD, Foster City, CA).
DNA Synthesis AssayThe full-length cDNA for murine stk (GenBankTM accession number X74736[GenBank]) was cloned by standard techniques and subcloned into the mammalian expression vector, pEV7. Tritiated thymidine uptake was measured in NIH 3T3 cells expressing stk as described (14).
We expressed human proMSP in CHO cells and activated the material
in vitro with kallikrein. The recombinant material had less than 10% of the specific activity of an MSP sample purified from bovine serum, as tested in a murine colonic crypt attachment
bioassay.2 Furthermore, active MSP from human plasma is
reported to be a disulfide-linked heterodimer, but after in
vitro kallikrein activation, the 80-kDa tertiary structure does
not remain intact. Subunits of our recombinant MSP were mostly not
disulfide linked, as judged by SDS-PAGE performed under nonreducing
conditions (Fig. 1, lane 4). Incorrect
proteolytic processing did not seem to account for this defect, because
the existence of the expected N-terminal sequence of the subunit,
VVGGHP, was confirmed by Edman N-terminal sequencing.
The lack of a disulfide bond between the subunits of recombinant MSP
suggested that the Cys content of MSP should be examined for a possible
cause of this defect and the low activity of our recombinant
preparation. The domain and disulfide structures of murine and human
plasminogen-related growth factors were first studied with respect to
the kringle domains (15). Later the sequences of MSP from chicken (16)
and MSP from rat (17) became available. These sequences confirmed that
the domain and disulfide structure is highly homologous to that of
plasminogen (Fig. 2). From the N terminus, the domain
structure of plasminogen may be summarized to contain a secretion
signal peptide, an N-terminal "hairpin" domain, five kringle
domains, and the SPD. The domain structures of MSP and HGF are very
similar to that of plasminogen. The main difference is that MSP and HGF
have only four kringles. The disulfide structure of plasminogen
contains intra- and interdomain disulfide bonds. The intradomain
disulfide bonds may be listed as follows: the hairpin domain contains
two disulfides, each kringle contains three disulfides, and the SPD
contains four disulfides. There is an interdomain disulfide between the
second and third kringle, and two disulfides between the subunit
and the SPD. According to conserved Cys content, every disulfide that
is present in plasminogen is also present in HGF and MSP, with one
exception: HGF and MSP have only a single disulfide between the
subunit and the SPD (15). HGF from chicken, mouse, and human contain no
Cys other than those that are present at positions that are homologous
to plasminogen (16). MSP, however contains extra Cys, some of which are
conserved in chicken, mouse, rat, and human (16, 17). Thus, murine MSP
has a Cys residue in the signal sequence, and murine and human MSP
contain an extra Cys in the hairpin domain. MSP from all four species
contain three extra conserved Cys in the SPD compared with both
plasminogen and HGF (Figs. 2 and 3). Because HGF does
not contain extra Cys compared with plasminogen and recombinant HGF of
high specific activity is available commercially, we considered whether
the extra conserved Cys residues of MSP caused the disulfide bonding
defect and low activity that we observed with our recombinant MSP
preparation.
Although high resolution structural information is not available for
plasminogen, MSP, or HGF, this information is available for other
serine proteinases, such as trypsin (18). Fig. 3 shows a sequence
alignment of the SPDs of MSP, plasminogen, and mature human trypsin.
The trypsin residues that were selected as homologous to the extra Cys
of the MSP SPD are numbered3 in Fig. 3 and
labeled in Fig. 4. Trypsin has ten Cys residues that
form five disulfide bonds, of which four have homologs in MSP (and
plasminogen). We used the structure of
diisopropylfluorophosphate-inhibited human trypsin described in the
Brookhaven file, 1trn, for our modeling. As can be seen in Fig. 4,
Lys66 and Arg95 are located on the surface of
trypsin on the opposite side of the protein from the
Ser127, the homolog of the Cys that forms the
intersubunit disulfide. Gln209 and Ser127 are
located on the surface in very close proximity. The distance between
the carbons of these residues is 0.61 nm. For comparison, the
distances between the
carbons of the disulfide-bonded Cys residues
of trypsin range from 0.42 to 0.62 nm. Thus, the inferred close
proximity of Cys672 to Cys588 suggests that
intrasubunit disulfide formation between these two Cys residues might
interfere with intersubunit disulfide formation by Cys588.
It should be noted that Cys672 apparently has no Cys
residue other than Cys588 with which to interact.
Cys672 of human MSP was mutated to Ala to determine if this
would eliminate the proposed intrasubunit disulfide bond. The mutated MSP was expressed, purified, and activated by kallikrein. The Cys672 Ala mutant MSP has subunits that are fully
disulfide linked (Fig. 1, lane 8) and has significantly more
activity than the nonmutant MSP, as judged in a tritiated thymidine
uptake assay in NIH 3T3 cells that are expressing transfected murine
stk gene (Fig. 5).
Wang et al. have reported production of recombinant kallikrein-activated MSP whose activity is comparable with that of serum MSP (10, 19). However, these studies did not address the occurrence of nondisulfide bonded (inactive) material because the recombinant MSP was not analyzed by nonreduced SDS-PAGE.
The Cys residue corresponding to human Cys672 is conserved in every species from which MSP has been cloned, and this deserves comment for two reasons. Firstly, the presence of Cys672 may result in low specific activity material when MSP from these species is produced recombinantly. Murine recombinant MSP also has the disulfide bonding defect that is described for human MSP in this paper and that is corrected by an analogous Cys to Ala mutation.4 Although chicken MSP has been cloned (16), there are no published reports on the recombinant expression of chicken MSP. This may be due, in part, to the complication of low activity described here for recombinant human or murine MSP. Secondly, the conservation of Cys672 suggests that the phenomenon described here may have a biological role in the regulation of MSP activity in vivo. In other words, circulating natural proMSP may mimic recombinant MSP by lacking the disulfide bond between the nascent subunits, and activation of proMSP in vivo may require both proteolysis as well as disulfide formation or isomerization to achieve its active conformation.
The Cys672 Ala MSP described here allows for the
production of highly active preparations of recombinant MSP that will
be useful in studies to elucidate the biological role of MSP.