THEJOURNALOF

Vol. 268, No. 21. Issue of July 25, pp. 15461-15468,1993

Printed in U.S.A.

BIOLOGICAL CHEMISTRY

Cloning, Sequencing, and Expression of Human Macrophage

Stimulating Protein(MSP, MST1) Confirms MSP as a Member of the
Locates the MSP Gene on
Family of Kringle Proteins and
Chromosome 3*
(Received for publication, January 29, 1993, and in revised form, March 29, 1993)

Teizo YoshimuraS$,Naoya Yuhkill, Ming-Hai Wang$, AlisonSkeel$, and EdwardJ. Leonard$

From the $Immunopathology Section, Laboratory of Immunobwlogy, and the BGenetics Section, Laboratory of Viral
Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

nally given to a human serum protein that makes resident

A human hepatoma (HepG2) cell line library was
screened withan oligonucleotide probe formacrophage peritoneal mouse macrophages capable of responding to the
stimulating protein (MSP) to clone an MSP cDNA. chemoattractant C5a (1, 2). We recently purified MSP to
Deduced sequences of isolated clones were compared homogeneity by immunoaffinity column and high performwith peptide fragment sequences of MSP. MSPScDNA ance liquid chromatography-ion exchange chromatography
encoded most of the known sequence of MSP except (3). By SDS-polyacrylamide gel electrophoresis, the molecular
for a small segment of the 6 end of the open reading mass of MSP was about 70 kDa; under reducing conditions,
frame. Consequently, a hybrid 2300-base pair cDNA MSP comprised (Y and @ chains of about 41 and 22 kDa. TWO
that encoded the complete MSP amino acid sequence partial sequences from a lysylendopeptidase digest of the MSP
2 clones. Culture fluid from
COSwas constructed from
7 cells transfected with this full-length
MSP cDNA had a chain had a highly conserved motif (NYCRNPD) found in
MSP biological activity, and the expressed MSP was the triple disulfide loop structure (kringle) of a family of
proteins that includes prothrombin (4),plasminogen (51, and
detected by immunoprecipitation with
antibody
against nativeMSP. The deduced amino acid sequence hepatocyte growth factor/scatter factor (6, 7). In analogy to
of MSP includes 4 kringle domains, which have been prothrombin or plasminogen, we postulated that MSP circulates in the
blood as pro-MSP,which becomes activated when
found in hepatocyte growth factor and several proteins
of the blood coagulation system. Among them, MSP has an Arg-Val bond is cleavedby a serine protease to make
the highest sequence similarity to hepatocyte growth separate a and /3 chains characteristic of this family.
factor (46% identity).The MSP cDNA hybridized
We now report the cloning of MSP cDNA from mRNA of
strongly tomRNA from liver, andto a lesser extent to a human hepatoma cell line, HepG2. The probe for the initial
mRNA from kidney and pancreas, suggesting that a screening of the cDNA library was an oligonucleotide mixture
cell type in the liver is the source of MSP. Several based on the sequence of an MSP peptide (BU-11, Ref. 3)
cloned and sequenced MSP cDNAs had insertions or that included the conserved kringle motif. Because of alterdeletions, suggesting that alternatively spliced MSP native splicing that resulted in insertions or deletions, it was
mRNAsmay occur. This was reflected in Northern necessary to isolate and sequence several cDNAs to deduce
blots probed with an MSP cDNA, which showed more
the complete open reading frame for MSP. A hybrid cDNA
than one mRNA species. Furthermore, although the
gene coding for MSP is on chromosome 3, the sequence that coded for complete MSP was then constructed andused
of one of the cDNAs was identical with a unique se- for transfection of COS-7 cells. Immunoprecipitation of the
quence in chromosome 1, indicating that there may be COS-7 cell product revealed protein bands of the expected
a family of MSP genes, located on chromosomes 3 and mass, and thesecreted MSP was biologically active. Sequence
andrestrictionsite
analyses show that the MSP gene is
1.
located on chromosome 3.
MATERIALS AND METHODS

Reagents-DMEM, L-glutamine, and gentamycin were from Quality Biological, Inc., Gaithersburg, MD. Cystine-free RPMI 1640 was
* The costs of publication of this article were defrayed in part by from Advanced Biotechnologies, Inc., Silver Spring, MD. FCS was
the payment of page charges. This article must therefore be hereby from HyClone Laboratories. Protein G-Sepharose was from Pharwere
marked aduertisement in accordance with 18 U.S.C. Section 1734 macia LKB Biotechnology Inc. -p[32P]ATP and ~-[~~S]cysteine
from Amersham. Restriction enzymes and reagents for cDNA prepsolely to indicate this fact.
The nucleotide seguence(s)reported in thispaper has been submitted aration were from Bethesda Research Laboratories. DNA sequencing
reagents were from United States Biochemical Corp. CX-[~~S]&TP
to the GenBankTM/EMBL Data Bank with accessionnumber(s)
were from Du Pont-New England Nuclear. XZAP I1 vector was from
L11924.
5 TO whom correspondence and reprint requests should be ad- Stratagene. Oligonucleotides were from Operon Technologies, Inc.,
Alameda, CA.
dressed Bldg. 560, Rm. 12-46, NCI-FCRDC, Frederick, MD 21702.
The abbreviations used are: MSP, macrophage stimulating proReagents for preparation of EIgMC3b included sheep blood, antitein; ElgMC3b, sheep erythrocytes with bound complement compo- coagulated with acid/citrate/dextrose; veronal-buffered saline with
nent C3b; HGF/SF, hepatocyte growth factor/scatter factor; MST1, 0.1% gelatin (VBS-gel): 0.14 M NaC1, 0.1 mM veronal, pH 7.4, 1 mM
locator term for MSP on chromosome 3; DMEM, Dulbeccos modified MgC12,0.15 mMCaC12; rabbit IgM anti-Forssman antibody, kindly
Eagles medium; FCS, fetal calf serum; VBS, veronal-buffered saline; supplied by Dr. Tibor Borsos; C5-deficient AKR mouseserum, stored
ELISA, enzyme-linked immunosorbent assay; kb, kilobase(s); bp, at -80 C; Dulbeccos modified Eagles medium (DMEM); and ambase pair(s).
monium chloride lysis buffer (0.16 M NH,Cl, 0.01 M KHC03, 0.001 M

Macrophage stimulating protein (MSP) is the name origi-

15461

Cloning of Human Macrophage Stimulating

Protein

15462

sequenced by the primer extension method (10).

Southern Blot Analysis-Southern blot analysis was performed as
described (8) in a 1%agarose gel with 10 pg of EcoRI-cleaved DNA
per lane. Filters were hybridized a t 40 "C overnight in 30% formamide,
5 X SSC, 5 X Denhardt's solution, 100 pg/ml sheared-denatured
MSPS :14
2203
salmon sperm DNA, 1%SDS, and 1 X IO6dpm/ml probe. Filters were
L
4
Mlul
washed twice with 2 X SSC, 1%SDS at room temperature for 15 min
Mlul
and 0.3 X SSC, 1%SDS at 50 "C for 30 min prior to autoradiographic
1777 1890
MSPl3B
exposure.
An
Northern Blot Analysis-Northern blot analysis of poly(A) RNA
was done by the glyoxal-dimethyl sulfoxide method in a 1.2% agarose
974
MSPl2A
gel with MSPS cDNA probe (8). Hybridization was as described for
n
A"
118 bp
Southern blot analysis except that the temperature was 50 "C. The
filters were washed in 2 X SSC twice at room temperature, then once
' 313 429
974
2181
MSP15A
in 0.1 X SSC at 65 "C. A human multiple tissue Northern blot was
4
A
4
An
purchased from Clontech.
R1
118 bP
Expression of MSP in COS-7 Cells-MSP cDNA, MSP9.13B, was
FIG. 1. Representative clones of MSP cDNAs. The top bar
excised from pBluescript by EcoRI digestion, then subcloned into an
represents the open reading frame for MSP, as determined by the
expression vector, pCAGGS ( l l ) , which was a generous gift from Dr.
sequence of the MSP9.13B hybrid cDNA. The unhatched portion
J. Miyazaki, Tokyo University, Japan. Transfection of COS-7 cells
indicates a likely signal peptide (see Fig. 2). MSPS, the first 13 bpof
was carried out with N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethythe MSP coding sequence are deleted from this cDNA. Arrows show
lammonium methylsulfate (Boehringer Mannheim) according to inMluI restriction sites, used for preparing a fragment for insertion
structions of the supplier. Briefly, COS-7 cells were cultured on 100into the corresponding locus of MSP cDNA 13B. MSP13B, this
cDNA begins with bp -17 (counting the first base pair of the putative mm tissue culture dishes in 10 ml of DMEM containing 10% FCS.
80% confluent, medium was aspirated, and
methionine initiator codon as number 1). Compared with MSP9, base When cells became about
14 ml of fresh medium containing complexes of DNA and N-[1-(2,3pairs 1778-1889 are deleted. MSP12A, the sequence begins at bp -6;
dioleoy1oxy)propylj-N,N,N-trimethylammonium
methylsulfate was
triangle indicates insertion of the 8th intron (see "Discussion") into
added (10 pgof DNA and 70 pl of N-[l-(2,3-dioleoyloxy)propyl]the cDNA. M S P l M , begins at bp -25. The 4th exon (see "Discussion") is deleted. A single base pair substitutionthat creates anEcoRI N,N,N-trimethylammonium methylsulfate per plate). After overnight
site is shown at the arrow. Insertion of the 8th intron also occurs culture, medium was aspirated, and 30 ml offresh medium was added.
(triangle). The sequence after 2181 is totally different from that of Two to four days later, medium was collected and frozen. For metathe other clones and has a different chromosomal origin as shown in bolic labeling of human MSP, transfection was carried out in a 35mm-diameter well (CLUSTER6, Costar, Cambridge, MA) in 6 mlof
Fig. 9.
medium. The cells were then incubated in 1 ml of cystine-free RPMI
1640 supplemented with 2 mM glutamine, 10% FCS dialyzed against
i n e24 h.
EDTA, pH 7.4). EIgMC3b were prepared as follows: sheep erythro- phosphate-buffered saline, 250 pCi of ~ - [ ~ ~ S ] c y s t efor
Immunoprecipitation and Gel Electrophoresis-One-ml samples
cytes were washed twicewith VBS-gel and suspended to a concentration of 2 X 10' erythrocytes/ml. To 1 ml of erythrocytes were added from the metabolic labeling experiment were diluted with an equal
0.1 mlofIgM anti-Forssman antibody. After 45 min at 37 "C, the volume of 0.1 M Tris-HCI, pH 8.6,0.15 M NaCl, 0.5% Tween 20,0.1%
cells were washed and resuspended in 10 ml of VBS-gel + 0.6 ml of bovine serum albumin (wash buffer), and incubated with an approxAKR mouse serum. After 20 min at 37 "C, the cells were washed 3 imately 20-p1 packed volume of protein G-Sepharose for 1 h at 4 "C.
After a brief centrifugation in a Microfuge, supernatants were mixed
times in cold VBS-gel and then stored at 4 "C in 4 ml of VBS-gel.
with 20 pg of normal rabbit IgG and 20 pl of protein G-Sepharose.
Construction of cL3NA Librav, Screening, and DNA SequencingHepG2 cells were grown to confluence in RPMI 1640 containing 10% After incubation for another hour a t 4 "C,supernatants were obtained
FCS, and total RNA was isolated by a guanidinium isothiocyanate by centrifugation. Meanwhile, 20-pg quantities of normal rabbit IgG
method; poly(A) RNA was isolated by oligo(dT)-cellulose chromatog- and polyclonal anti-human MSP (3) werecoupled with 20 pl of
raphy (8). cDNAs were synthesized by random priming or oligo(dT) protein G-Sepharose by incubation in wash buffer without bovine
priming, then ligated to the XZAP I1 vector (9) to prepare libraries. serum albumin for 2 h at 4 "C. IgG-coupledprotein G-Sepharose was
An oligodeoxynucleotideprobe was prepared from a portionof human washed with wash buffer three times, then incubated with pretreated
MSP amino acid sequence [DDNYCRNPDG] previously reported samples (1 ml) for 1 h at 4 "C. After three washes with wash buffer
(Ref. 3, sequence BU 11).The sequence of the probe is shown below. and two washes with wash buffer without bovine serum albumin,
pellets were mixed with 40 p1 of double-strength sample buffer (20%
T
A
A
A
glycerol, 6% SDS, 10% 2-mercaptoethanol), boiled, and applied to a
5'-CC: TCI GGA TTI C I CAG TAG T$ TGG TC-3'
G
G
12.5% polyacrylamide gel. After electrophoresis at 13 mA for 4 h,the
Approximately 5 X 10' recombinant phages from the cDNA library gelwas stained by Coomassie Blue, treated with Enlightning (Du
constructed by random priming were screened by high density plaque Pont-New England Nuclear), dried, and exposed to Kodak XR film
hybridization with the 32P-labeledoligonucleotide probe. Hybridiza- at -80 "C.
Detection of Protein-bound Carbohydrate-SDS-polyacrylamide gel
tion to nitrocellulose filters was carried out overnight a t 45 " C in a
electrophoresis of pure natural MSP on a Pharmacia-LKB PhastGel
solution containing 6 X standard saline citrate (SSC),5 X Denhardt's
solution, 0.05% sodium pyrophosphate, 1%SDS, 100 pg/ml heat- apparatus was followed by transfer to nitrocellulose. Protein-bound
denatured, sheared, salmon sperm DNA, and 1 X lo6 dpm/ml probe. carbohydrate was detected with a GlycoTrack kit (Oxford G b o Filters were washed three times with 6 X SSC, 0.1% SDS a t 40 "C for Systems, Rosedale, NY) according to the directions of the manufac30 min and were dried and exposed overnight to XAR-5 films (Kodak) turer. Carbohydrate is oxidized with periodate, and the aldehyde
with two intensifying screens for 2 days a t -80 "C. Phagemids carried groups formed are reacted with biotin hydrazide. Bound biotin is
within XZAPI1 recombinants were rescued with helper phage (9). detected with a streptavidin-alkaline phosphatasereagent.
Measurement of EigMC3b Binding and Phagocytosi by Resident
cDNA inserts were subcloned into M13mp18, and single strands were
Mouse Peritoneal Macrophages-For phagocytosis experiments (3),
sequenced by the dideoxynucleoside triphosphate chain termination
0.5-ml aliquots of mouse resident peritoneal cells at a concentration
method (10).
A 295-base pair cDNA obtained from the first screening was used of 8 x los cells/ml of DMEM without FCS (obtained as described for
to clone a full-length cDNA from the cDNA library constructed by the MSPbioassay) were added to 24-well polystyrene plates (Costar).
oligo(dT) priming. Approximately 2 million phages were screened. Macrophages comprised about 70% of total peritoneal cells. After 1
h at 37 "C, the plates were washed twice with DMEM, and then 0.5
Filters were hybridized in a solution containing 30% formamide, 5 X
SSC, 5 X Denhardt's solution, 1%SDS, 100 pg/ml salmon sperm ml of EIgMC3b was added, followedby 0.1 mlof DMEM with
DNA, and 5 X lo6dpm/ml probe. After overnight incubation a t 50 "C, different concentrations of MSP. The ratio of erythrocytes to perifilters were washed three times at 50'C in 2 X SSC, 0.1% SDS toneal cells was 50:l. After incubation for specified intervals, nonsolution, for 30 min. Denatured double-stranded cDNA inserts were adherent EIgMC3b were washed out of the wells, and ammonium

Cloning of Human Macrophage Stimulating

Protein

15463
-10

-1

AGCCAGAAGG
1
1
90
ATG GGG TGG CTC CCA CTC
CTG CTG CTT CTG ACT CAA TGC GGG
TTAGTC CCTGGG CAG CGC TCG
CCA TTG AAT
GAC TTC CAA GTG CTC
CGG
Met Gly Trp Leu
Pro Leu LEU Leu Leu Leu Thr Gln CysLeu Gly Val Pro Gly Gln Arg Ser Pro Leu Asn Asp Phe Gln

Val

Leu

Arg

180
GGC

Gly

ACA GAG CTA CAG CAC CTG

ThrG1u Leu Gln His Leu

CTA CAT
GCG GTG GTG CCC
GGG CCT TGG CAG GAGGAT GTG GCA GAT GCT GAA
GAG TGT GCT GGT CGC TGT
LeuHis Ala Val Val Pro Gly Pro Trp Gln Glu Asp Val Ala Glu
AspCysAlaAlaGluGly Arg Cys
270

TTA ATG GAC CGG

TGCGCC TTC CAC TAC AAC GTG AGC GGT
AGC TGC
CAT
CAA CTGCTG CCA TGG ACT CAA
CAC TCG CCC CAC ACG
Gly Pro LEU Met AspCys Arg AlaPhe His Tyr Asn Val Ser Ser His Gly CysGln Leu Leu Pro Trp Thr
Gln His Ser ProHis Thr
GGG CCC

360

TTC AAG AAA GAC TAC GTA CGG TGC

ACC ATC ATG AAC AAT
GGG GTT GGG TAC CGGGGC
AGG CTG CGGCGT TCT GGG CGC TGT GAC CTC CAG
Arg Leu Arg Arg
Ser Gly Arg Cys AspLeu Phe Gln Lys Lys Asp Tyr Arg
Val ThrCys Ile Met hsn Asn Val
GlyGly Tyr Arg Gly
450

CCG TTC
AAT GAT CACAAG TAC ACG
CCC ACT CTC CGG
AAT
ACC ATG GCC ACG ACC GGT
GTGGGC CTG CCC TGC CAG GCT TGG AGC CAC AAG
Thr Met Ala Thr Thr Val GlyLeuGly
Pro Cys Gln Ala Trp SerHis Lys Phe Pro Asn His
Asp LYs Tyr Thr Pro Thr Leu Arg Asn
540

CTG GAA GAG AAC TGC

TTC CGT AAC CCT GAT
GGC GAC CCC GGA
GGT CCT TGG TGC TAC ACA ACA GAC GCT
CCTGTG CGCTTC CAG AGC
Gly Leu Glu Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Pro Gly Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ala Val Arg P

GGC

630

TGC G K ATC AAA TCC TGC CGG

GAGGCC GCG TGT GTCTGG TGC AAT GGC GAG GAA TAC CGC
GGC GCG GTA GAC CGC ACG GAG TCA
GGG CGC
Cys Gly11s Lys Ser Cys ArR Glu Ala Ala Cys Val TrD CYS Asn Glv Glu Flu Tvr Arg Gly Ala Val Ser
Asp Gly
Arg Arg
Thr Glu
720

GAG rGc CAG CGC TGG GAT CTT CAG CAC CCG CAC CAGCAC ccc TTC GAG CCG GGC AAG TTC CTC GAC CAA GGT CTG GAC GAC AAC TAT TGC
Glu Cys Gln Arg Trp Asp Leu
His Gln
Pro His Gln His Pro Phe Glu Pro Gly Lys Phe Leu ASD Gln Glv Leu Tvr
ASD CYS
ASP Asn
810
GG TCC GAG CGG CCA TGG TGC TAC ACT ACG GAT CCG CAGGAG
ATC
TTC GAG
TGT CGA
GAC CTC CCC TGC
CGCGGG TCC
CGG AAT CCT GAC
Arn Asn Pro ASP Glv Ser Glu Arg Pro Trp Cys Tyr Thr Thr Asp Pro Gln Ile Glu Arg Glu Phe Cys Asp Leu Ser
Pro Arg Cys Gly

900

GAG GCA CAG CCC CGC CAA GAG GCC ACA ACT GTC AGCGGG
TGC
AAG TTC
GGT GAG
CGC GGC TAC CGGGGC ACA GCC AAT ACC ACC ACT GCG
Glu Ala Gln Pro Arg
Gln Glu Ala Thr Thr Val Ser Cys Phe Arg Gly Lys Gly Glu Glv Tyr Arn Gly Thr Ala Asn Thr Thr Thr Ala
990
GGC GTA CCTTGC CAG CGT TGG GAC GCG CAA ATCCCG CAT CAG CAC CGA TTT ACG AAA
CCA TAC
GAA GCG TGC
AAA GAC CTT CGG AAC
GAG
Gly Val Pro Cvs ArK
Gln Tru Asu Ala
Gln Ile ProHis Gln His Arg Phe Thr Pro
Glu Lys Tyr AlaCys LysASP Leu ArK Glu Asn
1080
TGC TTC ACA CTG CGG GGC
CCC ATG CGC GCG GCC TTT TGC TAC CAG ATC CGG CGT
TTC TGC CGG AAC CCC GGC
GACTCA GAG GCG CCC TGG
Phe CysA m Asn Pro ASD Glv Ser Glu Ala Pro Trp Cys Phe Thr Leu Arg Pro Gly Met Arg Ala Ala Phe Cys Tyr Gln Ile

1170

TGT ACA GAC GAC GTG CGG CCC

GAC TGC
CAG TAC CACGG GCA GGG GAG CAGTAC CGC GGC ACG GTC AGC AAG ACC CGC AAG GGT GTC CAG
Cya Thr Asp Asp
Val Arg Pro Gln Asp CysHisTyr
Gly Ala Gly Glu Gln Tyr Arg Gly Ser
Thr Lys
Val Thr Arg Lys G1v Val Gln
1260

TGC GAG
CGG GAG
TGC CAG CGC TGG GCT
TCC GAG ACG CCG CAC AAG CCG CAG TTC ACG TTT ACC TCC FAA CCG CAT GCA CAA CTG
CYS GlnArn Tru Ser AlaGlu Thr Pro His LYS Pro Gln
Phe Thr Phe Thr
Ser Glu ProHis Ala Gln Leu
Glu Glu Asn Phe Cys Arg

AAC

TTC

1350

GGG GAT AGC CATGGG CCC TGG TGC TAC

ACG ATG GAC CCA AGG ACC CCA TTC GAC TAC TGT GCC CTG
TGC GCT
CGAGAT
CGC
AAC CCA GAT
Asn Pro Asp Gly Asp His
SerGly Pro Trp Cys Tyr Thr Met Asp Pro Arg Thr Pro Phe Asp Tyr Cys Ala Leu Arg Arg Cys Ala As
1440

GAC CAG CCG CCA TCA ATC CTG GAC CCC CCA GAC CAG GTG CAG GGC
TTTAAGGAGAGGAAGGTGTGTGAT CGG CTG GAT CAG CGG CGT TCC
Asp Gln Pro Pro Ser
I l e Leu Asp Pro Pro Asp Gln Val Gln Phe Glu Lys Cys Gly Lys Arg Val Asp Arg Leu Asp Gln Arg Arg
1530

CAG CAG
CAT TTC TGC
GGG GGG TCT
AAG CTGCGC GTG GTTGGG GGC CAT CCG GGC AAC TCA CCC TGG ACA GTC AGC TTG CGG AATGGCCGG
Lys Leu Arg Val Val GlyHisGlv
Pro Glv AsnSer Pro TTP Thr Val Ser Leu Arg Asn ArK Gln Gly
His Phe
Gln Cys Gly Gly Ser
t

1620

CAGTTC TCC TCC

TGC CAT ATG CCT CTC GGC
ACG TAT GAG GTA TGG TTG GGC ACC
CTA GTG AAG GAG TGG
CAG ATA CTG ACT GCC CGG TGC
Leu Val Lys Glu Gln Trp I1e Leu Thr Ala Arg Gln
Ser Ser
CysCys
Phe
His Met ProLeu Thr Gly Tyr Glu Val Trp Leu Glv Thr
1710
GGC TCC CAG CTT GTC
CTG TTC CAG AAC CAG
CCACAT GGA GAG CCA AGC CTA CAG CGG GTC CCA GTA GCC AAG GGG
ATG CCC
GTG TCA
TGT
Ser Cys
Gly Ser
GlyGln
ProLeu Val
Leu Phe Gln Asn Pro His
GlnGlv Glu Pro Ser Leu Gln Are Val Pro Val Ala Lys Met Val
1800

CCA ACC
CTG CTC AAG CTG GAG AGA TCT GTG ACC CTG AAC CAG CGT GTG GCC CTG ATC TGG
TGC TAT
CTG GTG
CCC GTG
CCT CCT
GAAGGG
Leu Leu Lys Leu Glu ArK Ser Val Thr Leu A Asn
m ValGlnAla Leu Ile Cys Leu Pro Pro
TrpGLu
Tyr Val Val pro Pro Gly Thr
1890

AAG TGT GAG ATT GGC

GCA TGG GGT GAG ACC AAA GGT ACG GGT AAT GAC ACA GTC CTA AAT GTG GCC TTT CTG AAT GTT ATC TCC
Lys CYS Flu Ile Ala Gly TrD Gly Glu Thr Lys Gly Thr Gly Asn Asp Thr Val Leu Asn Val Ala
110 Leu
Ser Asn
Leu~ Asn
l n Val

AAC

1980

TGT GCC
GAG GGT
GAG TGT AAC ATC AAG CAC CGA GGA CGT GTG CGG GAG AGT GAG ATG TGC ACT GAG GGA
GTG GGG
CTGGCc
TTG
CCT GAC
Glu Cys Asn Ile His
Lys Arg Gly ArgV a l Arg Glu Ser Glu Met Cys Thr Glu Gly Leu Leu Ala Pro Val Fly Ala ASP
Cy5 Glu Gly
2070

TAC GGG GGC CCA CTT GCC

TGC TTT ACC CAC AACTGC TGG GTC CTGGAA GGA ATT ATA ATC CCC AAC CGA
TGC GTA
GCA A= TCC CGC TGG
Tyr Gly Gly Pro
Leu Ala CYS Phe Thr
His Asn CYS Trp Val
Leu Glu Gly Ile Ile Ile Pro Asn Arg Val Cys
SerAla
Arg Arg
Trp
2167

GCCCAGCCTTGATGCCATATGCCTTGGGGAG
CCA GCT GTC TTC ACG CGT GTC TCT GTG TTT GTG GAC TGG ATT CAC AAG GTC ATG
AGA CTG GGT TAG
Pro Ala Val
Phe Thr Arg Val Ser Val Phe Val Asp Trp
His Lys
Ile ValM e t Arg Leu GlyEnd
2200

GACAAAACTTCTTGTCAGACATAAAGCCATGTTTCCTCTTT(A),,

FIG. 2. NucIeotide and deduced amino sequences of human MSP. The arrows indicate a possible signal peptide cleavage site (1.) and
the cleavage site between the a and p chains
Underlined segments show sequences obtained for peptLdes of a lysylpeptidase digest of
human MSP (3). These sequence data are available from EMBL/GenBank/DDBJ under accession number L11924.

(t).

chloride buffer was added to lyse adherenterythrocytes without

affecting ingested erythrocytes. This buffer was removed after 2.5
min, and the monolayers were stained with Diff-Quik. The bottoms
of the wells were then cut out and mounted on glass slides so that
cells could be examined with a l O O X oil immersion objective. The
percentage of macrophages with at least 1 ingested erythrocyte was

determined. An estimate of the concentration of MSP in the COS-7

cell culture fluid was made by reference to a dose-response curve with
purified MSP.
Sandwich ELZSA for MSP-The assay was similar to other ELISAs developed in this laboratory (12) and utilized two of our antibodies to natural human MSP.The capture antibodywas IgGmonoclonal

CAG

Cloning of Human
Macrophage
Stimulating
Protein

15464
kDa

pure MSP is identified. A cleavage site between residues 18

and 19, making a secreted protein with a mass of 78 kDa,
would fit with the frequencies of the last 6 residues in signal
peptides described by Heijne (13), except for proline in posi97 - MSP
tion -2, which did not occur in Heijne's series of proteins. As
66established by our previous work, MSP belongs to the family
" M S P Uchain
of kringle proteins. The sequenceshows that MSP has 4
45 kringles in the N chain, and,like other members of the family,
an R-V bond is the junction between N and /3 chains, which
31 jchain
- MSP
are joined by an interchain disulfide (6). Predicted masses of
21the N and /3 chains of secreted MSP are 53 and 25 kDa,
respectively. Asparagines at positions 72 and 296 in the N
14FIG.3. Western blot showing glycosylation in both a and /3 chain and position 615 in the @ chain are within consensus
sequences for N-linked carbohydrate. Protein-bound carbochains of MSP.
hydrate in natural MSP was found in both chains (Fig. 3).
Among theproteins in the kringlefamily, thetranslated
mouse anti-MSP. Detection antihodv wasrahhit polyclonal IgG antiMSP. Hound rahhit antihody was detected with phosphatase-laheled sequence of the MSP9.13B cDNA is most similar to HGF/
goatanti-rahhit IgC (.Jackson ImmunoResearch Lahoratories, Inc.,
SF, as shown in Fig. 4 (45% amino acid sequence identity,
West Grove, PA). Cross-reactivity of the anti-MSP antibodies with
and an additional 17% conservative substitutions (14)).
other memhers of the family of kringle proteins had not heen checked
Fig. 5A shows the hybridization of MSPS cDNA to multiple
in detail at the time of this study.
sizes of mRNA derived from HepG2 cells. This is consistent
with the fact that different lengths of cDNAs were cloned
RESULTS
(Fig. 1) and appear toreflect alternative splicing of the MSP
cDNA Cloning-Based on the findings that partial amino gene (13). To study MSP gene expression, hybridization of
acid sequences obtained from purified MSP were similar to MSPS to mRNAs
from different human tissues
was examined.
those of hepatocyte growth factor/scatter factor (HGF/SF)
As shown in Fig. 5R, liver expressed the highest level of MSP
and that preliminary Northern blot analysis with an oligo- mRNA. The hybridization pattern was exactly the same as
nucleotideprobe showed a faintbandatabout
4kb, we for HepG2 cells, suggestingthat the same alternative
splicing
expected that the size of the mRNA for MSP might be large.
occurs in normal liver cells. In addition to liver, weak hybridTherefore, we construct.ed a cDNA library
by random priming.
ization was seen to mRNAsfrom kidney and pancreas.
About 5 X 10" phages were screened for MSP with an oligoExpression of M S P in COS-7 Cells-To confirm that the
nucleotide probe shownunder "Materials andMethods." After
hybrid cDNA, MSP9.13B, codes for biologically active MSP,
the first screening, two weakly positive spots were found on
we transfected COS-7 cells with MSP9.13B,which was ligated
the film. In a second screening of these two clones, a single
into the EcoRI site of the expression vector, pCAGGS, conisolated plaque was picked from each plate. Phagemids that
taining the @-actin promotor(11).The metabolically labeled
contained cDNAs were rescued and DNA sequencing was
MSP9.13B product expressed in COS-7 cells was immunopreperformed. One of two clones, designated MSP6, encoded a
(3). After
cipitated
with an antibody against natural MSP
fragment of the N chain of MSP (BU-11) that we previously
radioautography,
three
bands
were
observed
in
the
lane (Fig.
reported (3).
4 ) where the supernatant of pCAGGS.MSP9.13B6,
lane
To cloneafull-lengthcDNA,
we constructed asecond
cDNA library by oligo(dT) priming. Using MSP6 as a probe, transfected cells was treated with anti-MSPIgG. The 63-kDa
we screened approximately 2 x lo6 clones, and 85 cDNA and 30-kDa bandsmigrated to exactly the sameplaces where
clones were isolated, sized on agarose gels, and sequenced. purified natural MSP migrated on the same gel (data not
probably corresponds touncleaved
One of the clones, MSP9, was found to encode most of the shown). The 80-kDa band
amino acid sequencesobtained from apartial digest of purified pro-MSP (see introduction). Nospecific bands were observed
MSP reported previously. The matching sequences were in 9 in the other lanes. These results suggest that the transfected
COS-7 cells secreted pro-MSP, and that
some of the molecules
peptides of thepartial digest and includeda total of74
residues from the N chain and 65 residues from the @ chain. were cleaved by a serine protease from the cells or FCS to
However, MSPS lacked the 5' end of the open reading frame form the cy and @ chains of active MSP.
Culture fluids from MSP9.13B-transfectedCOS-7
cells
(Fig. 1). Several other cloneshad the 5' end of the open
reading frame, but had either a deletion or insertion which were also tested for MSP biological activity and for MSP
resulted in the creation of a termination codon. Three exam- protein by sandwich ELISA. The concentration of MSP in
culture fluids from two transfected preparations of COS-7
ples are shown in Fig. 1.
cells ranged from 16 to 45 ng/ml. As shown in Fig. 7 , culture
We thenconstructed ahybridcDNAclone,MSP9.13B,
fluid from the transfected cells stimulated mouse peritoneal
using MSP13R to supply the 5' segment that was missing
from MSPS and substituting theMluI fragment of MSPS for macrophages to phagocytize EIgMC3b. The magnitude of the
MluI fragment of MSP13 (Fig. 1). The substitution replaced response shown in Fig. 7 corresponds to a concentration of
the region of MSPl3B that contained adeletionwith
the 15 ng/ml MSP, as determinedin the same assay from a dosecorresponding intact region of MSPS and wasdesigned to response curve with pure MSP isolated from human serum.
complete the open reading frame for MSP. The nucleotide The result indicates thatMSP9.13B encodes biologically acand the translated amino acid sequences, starting with the tive MSP.
Hybridization of Human M S P cDNA to Genomic DNAs of
putative initiator methionine, is shown in Fig. 2. The open
reading frame codes for 711 amino acids, with a total mass of Different Species-As shown above, our assaysystemuses
80 kDa. There is a series of leucines near the N terminus, mouse peritoneal cells to detect human MSP activity, with
which probably represents part of the signal peptide for this an EDso of about 10"" M. This suggests that mouse and
secreted protein. The location of the signal peptide cleavage human MSP aresimilar. Therefore, we examined the hybridsite will remain in doubt until the blocked N terminus (3) of ization of human MSP cDNA to genomic DNAs of other
1

(Hgf)

B D D P QflSIV

EIM

T l ~ S E l S ( Y ]ML

I ~ H ~ T R~ B R
R W D LH P H Q H P F E P G K F

i: ~

(Msp)

C G I K S C R E A A C V W C N G E E Y R G A V D R T E S G R E C

(Hgf)
(MSP)

T K Q L R V V N G I P T R T N I G W M V S L R Y R NK.HICGGSLIKESWVLThRQCFPS
R S
- G U S PUTmNt_RjR(711; G Q i F G ] Q G l S i

zj:

(Hgf)

M G N E K C S Q H H R G K V T L N E S E I C A G A E K I G S G P C E G D Y G G P L V C E Q H K M R M

(MSP)

ISBQ

686
674

RlIF/GdHg-

E UI NK [ E ] R u -

- R [ a M O T EG LLA PV B A [ T l A a F

T UC BN V

728
711
FIG. 4. Amino acid sequence comparison of MSP and HGF.

sequence of MSP. After establishing overlapping sequences

of 2 clones, we made a hybrid cDNA that had the complete
MSP coding sequence. Transfection of COS-7 cells with this
cDNA resulted in secretion of MSP, assessed by three independent assays: immunoprecipitation, MSP sandwich ELISA,
DISCUSSION
MSP
and
bioassay.
We made a computer search for sequences similar to the
To select a cell line suitable for cloning an MSPCDNA, we c~~~ for MSP and its translated amino acid sequence (14).
a sandwich
to assay
fluids from Thesearch revealed thatHan et al. (15), by screening
a human
different human cell lines for secreted MSP. Among the cell genomic DNA library with a probe coding for the second
lines tested (B-celh glioma, bronchus, liver,osteosarcoma,
kringle domain in bovine prothrombin, identified a gene codfibroblast), only culture fluid from the human hepatomaline ing for a4-kringle protein that has considerablesequence
HepG2 reacted in the ELI%. Screening the HepG2 library similarity tohepatocyte growth factor.The gene comprises 18
with a probe designed from the partialsequence of pure MSP exons, separated by 17 introns. The sequence of the 18 exons
yielded many cDNAs, none of which had thecomplete coding is identical with the sequence of the MSP cDNA. This work
species. As expected, human MSPcDNA hybridized to mouse
genomic DNA fragments (Fig. 8). Human MSP cDNA also
hybridized to rabbit, guinea pig, mouse, and rat DNAs, but
definite hybridization was not seen to chicken DNA.

I:

Cloning of Human Macrophage

Stimulating
Protein

15466
e4

U
n
0)

I
kb
kb
9.5 7.5 -

9.5 7.5-

10

1.4 --

1.4 -

COS/
MSP

FIG.5. Expression of MSP mRNA in HepG2 hepatoma cells

( A ) and in different normal human tissues ( B ) .The probe was
MSPS cDNA.

9367-

)
"

COS

Medium

FIG.7. Stimulation of macrophage uptake of EIgMC3b by

culture fluids of COS-7 cells transfected with MSP9.13b
('OS/MCP,
cDNA. COS/MSP, cellstransfectedwithMSP9.13H.
cells transfected withan unrelated cDNA. COS, nontransfected cells.
Medium, DMEM. Culture fluids+ EIgMC3b were added to adherent
mousemacrophagesinpolysytrene
wells. Aftera1-hincubation,
nonadherent EIgMC3b were washed
off, adherent EIgMC3b were
lysed, and the macrophages were stained. The percentage of macrophages with 1 or more ingested erythrocytes was determined. Bars
show mean -C S.E. for triplicate wells. The COS/MSP response was
equivalent to the stimulating effectof 15 ng/ml pure natural MSP.

43 30 -

COS/
MCP

20 14 -

12.2 -

6.0 5.0 4.0 3.0 2.0

FIG.6. Immunoprecipitation of metabolically labeled MSP

expressed in COS-7 cells by anti-MSP antibody. Lane 1, control
supernatant with rabbit IgG; lane 2, control supernatant'with antiMSP-IgG; lane 3, pCAGGS.MSP9.13R-transfectedcell supernatant
with rabbit IgC; lane 4 , pCAGGS.MSP9.13B-transfectedcell super-

"

kb

1.6 -

1.0 -

natant with anti-MSP-lgG.

FIG.8. Hybridization of human MSPS cDNA to EcoRI-diand our own complement each other, since our dataassign a
function to the product of the gene described by Han et al. Rested genomic DNAs of different species.
(15), and theirwork describes the sequence of the MSPgene.
The genedescribed by Han et ~ l (15),
.
which our work Confirmatory evidence comes from sequencingdata of Welch
establishes as thegene for MSP, is located on chromosome 3. et al. (17), summarizedin Fig. 9. Whereas thesequence of the
The evidence can be summarizedas follows. Thehuman
MSP gene upstream fromnucleotide 4928 is common to
sequences of both chromosomes 1 and 3, identity with chrogenomic probe, H3H2, which identifiesalocus present on
both chromosomes 1 (DNF15S1) and 3p (DNF15S2) corremosome 1 abruptly ceases downstream from this position,
sponds to nucleotides 918-2868 of the MSP genomic DNA which rules out a chromosome 1 location for the MSP gene
sequence reported by Han et d . (15). Carritt et al. (16) have and confirms assignmentof the MSPgene to chromosome 3.
shown thatH3H2 hybridizes tothreeHindIIIrestriction
As noted under"Results" and Fig. 1, we isolated many MSP
cDNA clones that had either deletions or insertions, which
fragments of human DNA, 2.0,3.8, and 8.0 kb inlength.
Analysis of human-rodent chromosome hybrids assigned the resulted in shifts in the reading frame and the creation
of
2.0-kb fragment to chromosome 3; the larger fragments were stop codons. MSPl5A is of particular interest, because comfroma corresponding part of chromosome 1 (which has a parison with the data of Welch et ~ l (17)
. reveals two pieces
different location of one of the HindIII sites). Since the H3H2
of evidence that assign this clone to chromosome 1,not
hybridization locus on the MSP gene is the 2.0-kb HindIII chromosome 3. First, there is a single base pair substitution
fragment, it follows that the MSPgene is on chromosome 3. at nucleotide 787 of MSP cDNA 15A that creates an EcoRI

Cloning of Human Macrophage Stimulating

Protein
4457

MSP GENE

INTRON 17

[2181]
4928
'
"'
-

15467

5031

~-~

"I
L~
~

~~

->

DNF 15S2 ;
(CHR 3) j

cDNA 15A

An
stop

FIG. 9. Chromosomal location of the MSP gene and MSP cDNA 15A gene. Shading of bars indicates cDNA or exon. The top bar
represents part of the MSP gene in a region downstream from the H3H2 probe that defines DNF15 loci on chromosomes 1 and 3 (16).
Nucleotides are numbered according to the complete MSPgenomic DNA sequence of Han et al. (15). Nucleotide numbers of the MSP9.13R
cDNA are shown in parentheses (see Fig. 2). Regions of chromosomes 3 and 1 shown in this figure have been sequenced (17). Thebottom bar
represents part of the sequence of MSP cDNA 15A. The region of the MSP gene from 445'4' to 4928 has 96% sequence identity with the
indicated region of chromosome 1. The critical distinguishingregion is from nucleotides 4928 to 5031. This region of the MSPgene is identical
with the corresuondinereeion
.. of chromosome 3. but not chromosome 1. Conversely, this region of MSP cDNA 15A (slashes) is identical with
the correspondingregion of chromosome 1, but not chromosome 3.

.,

restriction site as shown in Fig. 1 (located in the 7th exon at gene allele, alteration of the first member of the pair having
nucleotide 2150 in the MSPgene sequence of Han et al. (15)). occurred earlier. Since the 3pdeletion is very large, involving
This site is absent in the H3H2 region of chromosome 3, but many genes, it is statistically unlikely that the MSPgene is
is present in the H3H2 region of chromosome 1 (Ref. 17 and the cancer suppressor gene. If it were, the Knudson theory
Fig. 2). Second, as shown in Fig. 9, thesequence of the 3' end predicts absent MSPexpression in the tumor.We found MSP
of MSP15A matches the corresponding sequence of chromo- mRNA in Northern blots of normal kidney. If expression is
some 1, not chromosome 3. In the view of Welch et al. (17), by the renal cell type that is tumorigenic, the finding of a
position 4928 in Fig. 9 is an "ancient junction" where about normal MSP gene sequence in the corresponding tumor cells
gene.
20 kb of chromosome 3 sequence to the left of this point was would rule out MSP as the cancer suppressor
We have described three in vitro effects of MSP on resident
transposedto chromosome 1. Thisaccounts forsequence
identity between the two chromosomes to theleft of position peritoneal macrophages of the mouse: 1) stimulation of che4928. Thus, it appears that the 3' end of MSP cDNA 15A motactic responsiveness to C5a (1,2); 2) stimulation of uptake
represents ancient chromosome 1 sequence, and the5' end is of C3bi-coated erythrocytes via the CR1 receptor of resident
from transposed and altered chromosome3. MSP15A is rep- macrophages (3); 3) appearanceof long cytoplasmicprocesses
resentative of several cDNA clones with similar3' sequences. and pinocytic vesicles when macrophages are plated in polyThe cloning of these cDNAs from the HepG2 cDNA library styrene tissue culture wells (1). These three effects have a
raises thepossibility that theremay bea family of MSP genes, motility response in common: the first is characterized by
translational movement, the second by membrane internaliwith representatives on chromosomes3 and 1.
In the normal human population, the
alleles on chromo- zation, the third by plasma membrane and cytoplasmic prosome 3pat thelocus defined by the H3H2 probe are frequentlytein realignment. Among the members of the kringle protein
heterozygous. The difference in thetwo allelescan be detected family, the MSPsequence is mostclosely related to HGF/SF.
mitogenic activity for hepatocytes, HGF/SF
by size differences(2.3 uersu 2.0 kb) in genomic HindIII In addition to its
causes
disruption
of epithelial cell junctions and an increase
restriction fragments to
which H3H2 hybridizes in a Southern
in contrast
blot. This is also the location of the MSP gene, and H3H2- in cellular motility.Thus, both MSP and HGF/SF,
to
their
relatives
with
proteolytic
activity,
have
a
role
in cell
defined heterozygosityof the gene will depend on the presence
motility. The rangeof target cells and thebiochemical mechor absenceof an HindIII restriction site
a t position 917 of the
anism of action remain tobe determined.
is
within the
Han et al. (15) MSP gene. Since this site located
first MSP intron, the heterozygosity should not affect the
Acknoruledgments-We thankMark Gunnel1 at the Biomedical
MSP coding sequence.
Supercomputer Center of the Frederick Cancer Research and DevelIt is now well established that there isa loss of one or the opment Center for searching the sequence data bank and doing the
other member of the allelic pair defined by H3H2 on chro- sequence between MSP and HGF/SF. We also thank Drs. William
mosome 3p in hereditary kidney cancer (18) and small cell Modi, Michael Lerman, and Berton Zbar for helpful discussions.
lung cancer cells (19). Therefore, these tumors are deficient
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S., Tanaka, S., Appella. E., and
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Leonard, E. (1991) J. Exp. Med. 173, 1227-1234
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representinactivation of the remaining cancer suppressor
in Proteases ond Hiologicol Control (Reich, E.. Rifkin, D. H..and Shaw,

15468

Cloning of Human Macrophage Stimulating

Protein

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