Beruflich Dokumente
Kultur Dokumente
BIOLOGICAL CHEMISTRY
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
15461
15462
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
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
360
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
GGC
630
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
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
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
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
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
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).
CAG
Cloning of Human
Macrophage
Stimulating
Protein
15464
kDa
(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.
I:
15466
e4
U
n
0)
I
kb
kb
9.5 7.5 -
9.5 7.5-
10
1.4 --
1.4 -
COS/
MSP
9367-
)
"
COS
Medium
43 30 -
COS/
MCP
20 14 -
12.2 -
"
kb
1.6 -
1.0 -
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
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
REFERENCES
in one of the alleles coding for MSP. According to the theory 1. Leonard, E. J., and Skeel, A. (1976) Exp. Cell Res. 102,434-438
2. Leonard, E. J., and Skeel, A. (1978) Exp. CellRes. 114, 117-12fi
proposed by Knudson (ZO), one mechanism leading todevel3. Skeel, A,, Yoshirnura.T.,Showalter,
S., Tanaka, S., Appella. E., and
opment of cancer requires inactivation of both alleles of a
Leonard, E. (1991) J. Exp. Med. 173, 1227-1234
4. Park, C. H.,and Tulinsky, A. (1986) Hiochernistp 25,3977-3982
cancer suppressorgene. Thus, the 3p deletions in cancer
cells
5. Magnusson, S..Petersen, T. E., Sottrup-densen, L.. and CIaeys. H.(197.5)
representinactivation of the remaining cancer suppressor
in Proteases ond Hiologicol Control (Reich, E.. Rifkin, D. H..and Shaw,
15468
E., eds) pp. 123-149, Cold Spring HarborLaboratory Press, Cold Spring
Shevach, E. M., and Stroher, W.,eds) Wiley Interscience, New York
Harbor, NY
13. Andreadis, A,, Gallego, M. E., and Nadal-Ginard, B. (1987) Annu. Reu. Cell
6. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T. Shimonishi, M., SugiBiol. 3 , 207-242
mura, A., Tashiro, K., and Shimizu, S. (1989) Nature 342,440-443
14. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 1 2 ,
7. Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987) Nature 3 2 7 ,
387-395
239-242
1.5~Han, S., Stuart, L. A., and Degen, S. J. F. (1991) Biochemistry 30,97688. Maniatis, T., Fritsch, E. J., and Sambrook, J. (1982) Molecular Closing:A
9780
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
16. Carritt, B., Welch, H. M., and Parry-Jones, N. J. (1986) Am. J. Hum.
Harbor, NY
Genet. 38,428-436
9. Short, J. M., Fernandez, J. M., Sorge, J. A., and Huse, W.D. (1988) Nucleic
17. Welch, H. M., Darby, J. K., Pilz, A. J., KO,C. M., and Carritt, B. (1989)
Acids Res. 16., 75-83
Genomics 6,423-430
. - -10. Sanger, F., Nicklen, S., and Coulson, R. (1977) Proc. Natl. Acad.Sei.
18. Zbar, B., Brauch, H., Talmadge, C., and Linehan, M. (1987) Nature 3 2 7 ,
U.S. A. 74,54-63
121-124
11. Yoshimura, T., Yuhki, N., Moore, S. K., Appella E., Letman, M. I., and
19. Brauch, H., Johnson, B., Hovis, J., Yano, T., Gazdar, A,, Pettengill, 0. S.,
Leonard, E.J. (1989) FEBS Lett. 244,487-493'
Graziano, S., Sorenson, G. D., Poiesz, B., Minna, J., Linehan, M., and
12. Leonard, E. J., Sylvester, I., and Yoshimura, T. (1991) in Current Protocols
Zbar, B. (1957) N. Engl. J. Med. 3 1 7 , 1109-1113
in Immunology (Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., 20. Knudson, A. G., Jr. (1986) Annu. Reu. Genet. 20,231-251