Macrophage capping protein (MCP) is a Ca2+-sensi- tive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin.
Vol . 267, No. 23. Issue of August 15, pp. 16545-16552,1992 Printed in U.S.A
THEJOURNALOF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Human Macrophage CappingProtein cDNA A UNIQUE MEMBEROF
THE GELSOLIN/VILLIN FAMILY EXPRESSED PRIMARILY IN MACROPHAGES* (Received for publication, April 16, 1992)
Guissou A.Dabiri8, ClarenceL. Young& Joel Rosenbloomll, and Frederick S. Southwick$(( From the $Graduate Groupin Cell Biology, the University of Pennsylvania,the llDepartment of Anatomy and Histology, the 19104, and the §Department of Medicine, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania Infectious Disease Section, the Uniuersity of Florida College of Medicine, Gainesville, Florida 32610
Macrophage capping protein (MCP)is a Ca2+-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA forMCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCPexpressed in Escherichia coli using a pETl2a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-170ofthetotalcytoplasmicprotein. Northern blot analysis of U937 and HL60cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and > 7 times initial levels, respectively. Human MCPdisplays a 93%amino acid sequenceidentity with two recently described mouse proteins, gCap39 and Mbhl. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocytedevelopment into macrophages indicate that MCP may play an important rolein macrophage function.
Macrophage locomotion, phagocytosis, and degranulation probably require regulation of actin filament length and concentration (1).When phagocytic cells are exposed to a chemoattractant or a phagocytic stimulus, the actin filament (Factin) content increases approximately 2-fold (2-4). Experimental evidence suggests that this rise in F-actin is a result of actin monomer assembly onto the fast growing or barbed ends (as defined by electron micrographs of actin filaments decorated with myosin heads) of actin filaments (4, 5). Accordingly, actin-binding proteins which block monomer incorporation at this endare likely to play an importantrole in regulating actin assembly associated with cell movement and * This research was funded by the RobertWood Foundation (toC. L. Y.) and by National Institutesof Health Grants R 0 1 AR20553 (to J. R.) and ROlAI23262 (to F. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. T h e nucleotide sequencefs)reported in this paper has been submitted totheGenBankTM/EMBLDataBankwith accession. number(s) M94345.
11 TOwhom all correspondence should be sent: Infectious Disease Section, University of Florida College ,of Medicine, Box 100277, Gainesville, FL 32610. Tel.: 904-392-4058; Fax: 904-392-6481.
shape change. In mammalian cells five proteins have been described which can perform this function. Two of these, gelsolin and villin, have molecular masses in the 90 kDa range (6). Inaddition to capping the barbed ends, these two proteins can also sever actin filaments. The cDNAs of both proteins have been cloned from human cells and found to have very similar deduced amino acid sequences (7, 8). Gelsolin has a very broad tissue distribution that includes phagocytic cells, platelets, fibroblasts, and many other nonmuscle cells (9). This protein is also found in smoothand skeletal muscle cells and with an added sequence is secreted by these cells into the plasma (10). The distribution of villin, on the other hand, is limited to organs of the gastrointestinal and urogenital tracts (11). A third protein, adseverin, has a lower moIecuIar mass, 74 kDa. Amino acid sequencing, proteolysis, and actin function studies indicate that adseverin is both structurally and functionally similar to villin and gelsolin (12). Like villin, its tissuedistribution is restricted, being found primarily in neural and endocrine tissues (13). The fourth mammalian barbed end blocking protein, Cap Z, is a heterodimeric protein with subunits of M, = 36,000 (a subunit)and 32,000 ( p subunit) which was originally purified from skeletal muscle (14). Like gelsolin, this protein is found in most nonmuscle and muscle cells (15). Unlike villin, gelsolin, and adseverin, this protein does not sever actin filaments. Comparison of the primary structure of this protein with those of villin, gelsolin, and adseverin reveals little similarity, indicating that this protein is not homologous to these proteins (15). The fifth mammalian protein in this functional class is macrophage capping protein (MCP)’ (16,17). This protein has a molecular mass of 41 kDa by SDS-PAGE and is functionally similar to Cap Z (i.e. it caps the barbed ends of actin filaments without severing them). MCP differs from Cap Z in one functional aspect: the ability of Cap Z to bind actin is unaffected by changes in Ca2+,whereas MCP, like gelsolin and villin, requires micromolar Ca2+to initially bind actin. The distribution of MCP in other cell types and tissues has not been systematically investigated. We have now cloned the cDNA for MCP from a human monocytic cell line U937 and expressed the humanprotein in Escherichia coli. Comparison of the derived amino acid sequence has revealed a marked similarityto theNHz-terminal halves of gelsolin and villin, indicating that MCPis a member of the gelsolin/villin family of actin-binding proteins. The primary structure is nearly identical to thatof a mouse protein called gCap39 or Mbhl. Recombinant MCP is functionally identical to the native rabbit alveolar macrophage protein The abbreviations used are: MCP, macrophage capping protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid kb, kilobaseb); PMA, phorbol 12-myristate 13-acetate.
16545
16546
Molecular Cloning of Human Macrophage Capping Protein
withrespect to Ca2+ sensitivityandthe ability to block monomer exchange at the barbed ends of actin filaments. Western blot analysis has demonstrated that unlikegelsolin and Cap Z, MCP has a limited distribution and is primarily found in macrophages. Likewise, Northern blot analysis revealsdetectable message only in macrophages, monocytes, and kidney tissue. A markedincrease in MCP message is observed when U937 and HL60 cells are differentiated into macrophage-like cells. These findings suggest that MCP is likely to play an important and unique role in macrophage function. EXPERIMENTALPROCEDURES
Preparation and Sequencing of MCP Peptides-MCP was purified from rabbit alveolar macrophages as described previously (16). Purity of the protein was > 90% as determined by laser densitometry of Coomassie Blue-stained SDS-PAGE. Thepurified protein was lyophilized and cleaved using cyanogen bromide (CNBr) at a final concentration of 5.5 mg/ml in 70% formic acid (18). The CNBr cleavage products were separated by hydrophobic chromatography (C18 column, Waters Associates, Milford, MA) using a 0-60% acetonitrile gradient. The amino acid sequences of two major peaks were carried out by Edman degradation. cDNA Cloning and Nucleotide Sequencing-A nonredundant oligonucleotide (see "Results"), later found to correspond to amino acid residues 236-248of the intact protein (Glu-Glu-Asp-Leu-Thr-AlaAsp-Gln-Thr-Asn-Ala-Gln-Ala), was synthesized (MilliGen/Biosearch 7500, Burlington, MA) based on the codons most frequently used in human proteins (19). The oligonucleotide was end labeled with T 4 polynucleotide kinase and [ Y - ~ ' P ] ~ A T(3,000 P Ci/mmol, Amersham Corp.) and used to screen a X g t l O undifferentiated U937 cell cDNA library (Clonetech Laboratories, Palo Alto, CA). Hybridization, washes, and autoradiography were performed using standard conditions (20). Positive clones, identified on duplicate filters, were plaque purified, and their cDNA inserts were subcloned into pUC19. The cDNA was sequenced by dideoxy chain termination of doublestranded plasmid DNA and a modified T7 DNA polymerase (Sequenase; U. s. Biochemical Corp.). The cDNA ends were sequenced using the universal and reverse primers, and the remaining regions of both strands were sequenced by primer walking using a series of specific 20-mer oligonucleotides. Expression and Purification of Recombinant MCP-Following insertion of an NdeI restriction site by the polymerase chain reaction (20)atthe ATG start site, humanMCP cDNA was cloned into pETl2a at the NdeI and Sal1 polylinker sites (Novagen, Madison, WI). Nucleic acid sequencing confirmed that the polymerase chain reaction product inserted in place of the first 109 bases of the native cDNA (inserted at the StuI site) faithfully reproduced the original sequence with the exception of the change from AGC to CAT at bases 47-49 just antecedent to the open reading frame (see "Results"). BL21 (DE3) transfected with the pETl2a constructwere grown in 250 ml of LB medium containing 50-100 pg/ml ampicillin at 37 "C to an AeO0 = 0.6 and induced by the addition of 0.4 mM isopropyl 1thio-0-D-galactopyranoside (21). Cells were harvested 5 h postinduction by centrifugation at 4,400 X g for 10 min. The resultant bacterial pellets were frozen at -20 "C and then diluted in 125 ml of lysis buffer (50 pg/ml lysozyme in 20 mM imidazole HCI, pH 8, 2 mM EGTA and 1 mM phenylmethylsulfonyl fluoride). After incubation at 30 "C for 15 min the samples were centrifuged at 100,000 X g for 1 h. The supernatant was diluted 1:1 with DEAE buffer (10 mM imidazole HC1, pH 8, 1 mM EGTA, 0.1 mM MgCI2)and loaded on a fast protein liquid chromatography DEAE ion exchange column (DEAE-5PN, 15 cm X 20 mm, Toyohaus, Philadelphia, PA). Following a 50-ml wash with DEAE buffer, the column was elutedwitha 0-0.09 M KC1 gradient over 15 min at a flow rate of 5 ml/min. Purity was assessed by Coomassie Blue-stained SDS-PAGE. Purified protein was immediately stored at -20 "C in 1:l volume of ethylene glycol. RNA Isolation andNorthern Blots-Total RNA was extracted according to a modified guanidinium thiocyanate method (22). RNA was separated on 1.2% agarose gels, transferred to 0.45-pm nitrocellulose paper (Schleicher & Schuell), and hybridized with 3ZP-labeled random primed MCP cDNA at 42 "C in 5 X Denhardt's, 5 X SSC, 50% formamide, 150 pg/ml salmonsperm DNA, and 1%dextran sulfate. A full-length cDNA for @-actin, cloned from human fibroblasts, was kindly provided by Dr. Larry Kedes (University of South-
ern California, Los Angeles, CA) (23). Gelsolin cDNA (a 1.9 kb of the 2.1-kb full-length gelsolin) was kindly provided by Dr. David Kwiatkowski (Harvard Medical School, Boston, MA). Genomic DNA Isolation andSouthern Blots-High molecular weight human genomic DNA was isolated from U937 cells, CCL202 fibroblasts, and human lymphocytes and digested with restriction enzymes according to standard methods (20). All DNA samples were subjected to electrophoresis on 0.7% agarose gels in TAE(Tris acetate/EDTA) or TBE (Tris borate/EDTA)(20) and transferred to supported nitrocellulose filters (Schleicher& Schuell). The blots were hybridized at 65 "C in 6 X SSC, 2 X Denhardt's and 100 pg/ml salmon sperm DNA using a 3ZP-labeled random primed 1.2-kb MCP cDNA fragment. The filters were washed in 6 X SSC, 2 X Denhardt's for 45 min at 65 "C, followed by a wash in 2 X SSC and 0.1 M sodium phophate for 20 min at 65 "C, and a final wash was at 65 "C in 0.5 X SSC and 0.1% SDS. Cell and Tissue Extract Preparation-Rabbit polymorphonuclear leukocytes, erythrocytes, lymphocytes, monocytes, platelets, and plasma were obtained by separation on Ficoll-Hypaque (Lymphocyte Resolving Medium, Flow Laboratories,McLean, VA) (24).Rabbit alveolar macrophages were harvested as described previously (16). Tissues were obtained from freshly killed rabbits andquickly homogenized at 4 "C. Cytoplasmic extracts were prepared using standard methods (9).The serineproteaseinhibitor diisopropylfluoropbosphate was used in all cases. Immunoblotting of Cell Extracts-Purified proteins orcell extracts were subjected to electrophoresis on 5-15% discontinuous slab SDSPAGE (26). The electropboresed.proteins were then transferred to nitrocellulose paper using the standard protocol for the MilliblotSDE ElectroblottingApparatus (Millipore Corporation, Bedford, MA) (27). The transferred blots were washed three times for 5 min each in phosphate-buffered saline serumand 0.3% Tween 20 followed by a blocking step with 10% normal goat serum for 30 min. The primary antibody, guinea pig anti-rabbit MCP polyclonal antibody, was diluted 1:500 in the blocking solution and incubated for 1 h at room temperature. After subsequent washes with phosphate-buffered saline/Tween, the blots were bathed in a 1:5,000 dilution of a biotinconjugated secondaryantibody (monoclonal anti-guinea pig IgG, Sigma) for 1 h, washed, and incubated with 1:10,000 avidin-alkaline phosphatase conjugate (extravidin,Sigma) for 1 h. The detection solution consisted of alkaline phosphatase buffer (0.1 M Tris, 5 mM MgCl,,0.1 M NaCI) containing 30 pg/ml nitro blue tetrazolium (Sigma) and 20 pg/ml5-bromo-4-chloro-3-indolyl phosphate (Sigma). Cell Culture and Induction of Differentiation-Monocyte-derived macrophages were obtained by culturing the cells in RPMI 1640 medium with 10% fetal calf serum and 1,000 units/ml macrophage colony-stimulating factor for 4 days. Macrophage colony-stimulating factor was added to enhance cell viability and yield. U937 cells and HL60 cells were maintained in RPMI 1640 medium containing 10% iron-supplemented calf serum and 2 mM glutamine. For macrophage differentiation, exponentially growing U937 and HL60 cells (>1 X lo6 cells/ml) were plated at 0.75 X lo6 cells/ml in 100-mm tissue culture Petri dishes (Falcon). Phorbal12-myristate13-acetate (PMA, a gift of Dan Liebermann, University of Pennsylvania, Philadelphia) dissolved at 0.1 pg/ml in dimethyl sulfoxide was used to treat U937 cells at 100 nM (62 ng/ml) and HL60 cells at 10 nM (6.2 ng/ml). Macrophage differentiation was assessed by increased adherence, morphological changes, loss of proliferative ability, and nonspecific esterase reaction (Sigma diagnostics kit 90-Al). At appropriate time pointsafter differentiation, RNA was extracted and analyzed on Northern blots as described earlier. Deglycosylation Experiments-For digestion with endoglycosidase F (Boehringer Mannheim), 100 pg of rabbit macrophage extract was incubated overnight with 0.6 unit of the enzyme in a total reaction volume of 10 p1 at 37 "C. The samples were frozen at -20 "C until loaded on SDS-PAGE. For digestion with 0-glycanase (Boehringer Mannheim), 10 pl of macrophage extract (100 pg) was incubated with 0.5 milliunits of the enzyme at 37 "C for 4-5 h before electrophoresis on SDS-PAGE. The migration of MCP was detected by immunoblotting. For endoglycosidase H (Boehringer Mannheim) digestion, 10 pg of purified MCP was incubated overnight with 10 milliunits of the enzyme in a 20-pl reaction volume at 37 "C. Migration was assessed by Coomassie Blue staining of SDS-PAGE. RESULTS
Isolation of cDNA Clones and Open Reading Frame Analysis-Two peptide fragments from CNBr digestion of rabbit
Protein
of Human Macrophage Capping
Molecular Cloning
16541
reading frame codes for a 348-residue protein with a predicted M , of 38,386. This molecular weight agreed reasonably well with those determined by SDS-PAGE and analytical gel filtration chromatography (MI 41,000). Similar discrepancies C T G A C A G C T G A T C A T A C C A A T G C T G A G G C T 3 ' ) between SDS-PAGE and amino acid sequence-derived molecwas synthesized and used to isolate clones for human MCP ular weights have also been observed for gelsolin (90,000 by froma X g t l O cDNA library derived from a U937 human SDS-PAGE versus 83,000 by sequence analysis) (7) andvillin monocytic cell line. This oligonucleotide probe was subse- (95,000 uersus 92,284) (29). Expression, Purification,and Functional Characterizationof quently found to correspond to bases 755-772 and to share 82% identity with the native sequence. Two positive clones Human MCP-The full-length open reading frame starting were present on duplicate filters from the 80,000 plaques at the first ATG site (bases 50-53)was inserted into the screened. The nucleotide sequence and thederived amino acid PET12a expression vector using the NdeI and Sal1 polylinker sequence for the plaque-purified clone are shown on Fig. 1. sites. An NdeI restriction sitewas introduced into MCP cDNA The sequence was 1,259 base pairs long, which corresponded by polymerase chain reaction to allow a restriction cut imto thesize of the mRNA (1.2 kb) identified by Northern blots mediately before the ATG start codon. After transfection into ofU937 total RNA preparations (see below, Fig. 5). The BL21 (DE5) E. coli strain, protein synthesis was induced by cDNA contained an open reading frame which started at the 0.4 mM isopropyl 1-thio-(3-D-galactopyranoside.The time first ATG codon at position 50 and ended with the TAG stop course of bacterial protein synthesis was monitored by SDScodon at position 1094followedby a polyadenylation site PAGE of whole bacteria. Maximum production was observed between bases 1200 and 1205. The initiatorcodon (ATG) was 5 h after induction. As compared with time 0, the concentrapreceded by a consensus sequence commonly associated with tion of the polypeptide increased nearly 20-fold. At this time the translationinitiation site. This sequence contained cyto- point the resultantrecombinant polypeptide represented 23sines at the -1 and the -4 positions and an adenine at the 25% of the total bacterial protein. Greater than 85% of this -3 position (28). Since the amino terminus was blocked, expressed protein was soluble in the lysis solution. Following identification of the first methionine residue could not be bacterial lysis the supernatantwas diluted with DEAE buffer verified by amino acid sequencing. Bacterial expression using and loaded on a DEAE ion exchange column. The recombithe predicted MCP cDNA open reading frame has, however, nant protein was found to weakly adhere to thecolumn while yielded a protein of size and activity similar to purified rabbit all the native E. coli proteins bound more tightly. Introduction MCP (see below). of low concentrations of KC1 (0.01 M) resulted in the elution Assuming initiation of translation at the first ATG codon of a single homogeneous peak of protein. A 250-ml culture of the predicted amino acid sequence contained the two se- bacteria yielded 12-15 mg of MCP. Purity of the protein was quences derived from the rabbit alveolar macrophage peptides greater than 99% as estimated by densitometry scanning of (underlined regions of Fig. 1).Three amino acid substitutions Coomassie Blue-stained SDS-PAGE. Within 5 h of bacterial were observed in the rabbit oligopeptide 1 as compared with lysis, highly purified recombinant protein was available for the human sequence (amino acid 229, Pro for Ala; amino acid functional studies. 243, Gln for Lys; and amino acid 244, Thr for Ala, hashed Activity of the recombinant proteinfrom the DEAE column underlines). One amino acid substitution was observed in fractions was initially assayed using the falling ball microvrabbit oligopeptide 2 (amino acid 321, Arg for Gln). These iscometric assay (30). Polymerization of 15 PM actin (final changes may be attributed to species differences. The open concentration) in a solution containing 0.2-0.4 PM recombimacrophage MCP were subjected to amino acid sequencing. From a region of low codon degeneracy in one of the peptide fragments(residues 236-248of peptide 1) (Fig. 1) a long nonredundant oligonucleotide (39 bases, 5' GAG GAG GAC
CGCAGGCTGGAAGGAAW\CGAACCTACGAAGCAGAGATCTGAAGACAGC
ATG TAC ACA GCC ATT CCC CAG GGC TCT AGT CCAT X CCA GGC TCA GTG M Y T A I P Q S G S P F P G S V GAG AAG CTG AAG CCG E K L K P V
CAG GAT GGC CCA CTG Q D P G L
CAT GTG TGG CGG H V W R
GTG CCT GTG CAA GAG GCG AAC CAG GGC GTC TTC TTC TCG GGG GAC TCC TAC CTA P V A Q E N Q G V F F S G D S Y L V
49
GTG 127 V 26
GTG CTG 205 CAC L H 52
AAT GGCCCA GAA GAG GTT K C CAT CTGCAC CTG TGG ATA GGCCAG CAG TCA K C CGG GAT GAGCAG GGG GCC TGT GCC 283 E V N G P E S H L H L W I G O Q s s R D E Q G A C A 18 CTG CTG GGA GAG CGG CCT GTG GTG CTGK T GTG CAC CTC AAC ACG V L A V H L N T L L G E R P V Q
FIG. 1. Nucleotide and deduced amino acid sequence of human MCP cDNA. Residue numbers are shown at the right. Sequences of peptides 1 and 2 (underlined) were obtained from Edman degradation of CNBr fragments of rabbit MCP. Amino acid substitutions in the rabbit MCP are indicated by hashed underlines. The bracketed sequence represents amino acids of low codon degeneracy from which the unique 39-mer oligonucleotide probe was synthesized.
CTC TTC ATG L F M S GGA
G
CAG CAC CGC GAG GTG CAG GGC AAT 361 GAG H R E V Q G N E S D 104
AGC TTC TACCCA CGGGGC CTC AAG TAC CAG GAA GGT GGT GTG TCA GAG GcI\ TTT CAC AAG Y F P R G L K Y Q E G G V E S A F H K T
GCC CCA GCT A P A A
GCC AAG ATC A h 4 CTC TAC CAG GTG I K K L Y Q V K
GG AAG G AAG
G
AAG AAC ATC K N I R
K
CGT GCC ACC A T E R
TCT
GAC
ACC TCC 439 ACA S T 130
GAG CGG GCA511 CTG A L N 156
AAC
TGG GACAGC TTC AAC ACTGGG GAC TGC TTC A N CTG GAC CTGGGC CAG AAC ATCTTC GCC TGG TGT GGT GGA AAG TCC 595 W D S F N T G D C F I L D L G Q N I F A W C G G K S 182 AAC ATC CTG GAA CGC AAC AAG N I L E R N K A GAG E
ATT I
GTC V
ACT
T
D
CCTGAG GAA GAC P
E
E
~
CTC I
ATG AAC CTG ACC M N L T K
GCG AGG GAC R D L A
GATGAGtGG GAG
CCT
G
P
E
E
ACA GCT GAC ,
T
A
GCA
D
~
AAG GTG GCT V A D S
GAGGGC TTC ATC TCG E G F I s ~
CGC n
E
AAG A
AAC tGG CTC TGTGGC AAG ATC TAT N G L C G K I Y I GCC
GCT A
M
ATG T
ATC O
N
A
O
ATC TTC AAG CAA TTT TTCAAG GAC TGG A T F K O F F K D W K
CAG TAC y ; & ; , ~ A
GTC
L
G A
CTG P
GCC)GCA A
GCT A
GGC
K
P
CTG L
Y
CCC GAA TTTCTGGCCCTGCTTATA TCT L E L L I S D
ATC TGGGGGAAG CGA AAA GCG W K G R K A -
GCC ATC CCG D S E R
CAG
V
Peptide &I AAT GCC CAG
GAC TCC AGC S P F A
ATG 9
GAG
CTG GCC CTG L A I R
GCC
CC6 T
0
AAT GAG AAG GAG N E K E AAC
v
ACT CAG E I L
GAC ACT GAG Q G K A CCC A
AAG L
p
CCT
K
E
GCT G
N
CTG151AAG
TAT TCT GAT AAG GCC GTC ACT GGA CAG K
V
~
D
GAT GAC TGC D C F V
CGG CAG GCA R Q A A GTG
CGA CAG 673 GGC Q V 208
A
GCC
GAG
GGC
234 829 T 260 G
TTT GTG 901 CTG L D 286
GCC CTG CAG985 GTG L Q V 312
GAGGGCATT CGT CTG GAG CCT AGT CAG CCC 1063 338 G R E s p
0
M TGRGGGTGGGCGTCTTCCTGCCCCATGCTCCCCTGCCCCCCACCACCTGCCTGCTTGCTTCTC 1156
'
AAG
348
T G G C T G C C T G G T C A G T C T G C C C C C T G C A G A T G T T C A A T A A A G G A G A C A A G T G C T T T C C C P 1259
O
GAC
Molecular Cloning
16548
of H u m a n Macrophage Capping Protein
nant protein (final concentration) was associated with a decrease in the viscosity of the solution to that of buffer when Ca2+was in the mM range. The addition of EGTA to reduce Ca2+ concentration to the nanomolar range totally reversed this effect, viscosity recovering to that of actin polymerized in buffer (data not shown). The ability of recombinant MCP t o block the barbed ends of actin filaments was measured using pyrenyl actin. The apparent capping constant was 0.20.4 nM as assessed by measuring the rates of pyrenyl actin filament disassembly in the presence of varying concentrations of recombinant MCP (see Fig. 2). The ability to block actin monomer exchange at the barbed end required Ca2+. End blocking activity was completely inhibited when Ca2+ was lowered to the nM range by the addition of EGTA (data not shown). Recombinant MCP also blocked actin assembly from actin-spectrinnuclei in a calcium-sensitive fashion(data not shown). Analysis of Glycosylation Sites-Others have suggested MCP may be actively secreted in cultured macrophages (31), a behavior which could be mediated by glycosylation; furthermore thepredicted amino acid sequencecontained two potential sites for N-linked glycosylation with the consensus sequence Asn-X-Ser at amino acid positions 101-103 and the sequence Asn-X-Thr atpositions 262-264 (Fig. 1). We therefore studied the effects of various glycosidases on MCP migration on SDS-PAGE. Exposure to endoglycosidase H had no affect on the migration of the purified intact protein on SDS-PAGE (data not shown).Also, the presence or absence of endoglycosidase F in macrophage extracts had noeffect on the migration characteristics of the 41-kDa band identified by immunoblotting(Fig. 3) (see “Tissue Distributionby Western and Northern Blots” for antibody specificity). Both of these enzymes are specific for N-glycans; however, they are each specific for different combinations of the sugar groups. T h e possibility of the presence of 0-linked glycosylation sites was also examined. As seen on Fig. 3, a 41-kDa band was detected in the presence and absence of 0-glycanase.In addition, the protein failed to take up periodic acid Schiff stain, a colorimetric assay for highly glycosylated proteins (data not shown). The bacterially expressed protein, which would not be expected to be glycosylated, has actin binding properties similar to those of the purified native protein, indicating that MCPprobably does not require glycosylation for its interactionwith actin. Computer Search Analysis-MCP was found to have a high
0-glycanase
97 68 43
- c
-”
t l81
L
I
I
43
29
I
FIG. 3. Digestion of MCP with endoglycosidase F and 0glycanase. A 41-kDa band is present in thepresence (+) and absence (-) of either enzyme. 100 rg of rabbit macrophage extract was digested with endoglycosidase F for digestion of N-linked sugars and 0-glycanase for 0-linked sugars. Migrationof MCP was detected by immunoblotting (see “Experimental Procedures”). HCP (H)
KYTAIPQSGSPFPGSVQDPGLHVWRVEKLKPVPVAQENQGVFFSGDSYLV I I I I I II I
50
g ~ a p 3 9 ~ 1s
MYTPIPQSGSPFPASVQDPGLHIWKLKPVPIARESHGIFFSGDSYLV
50
Kbhl(t4)
1
MYTPIPQSGSPLPASVQDPGLHIPKLKPVPIARESHGIFFSGDSYLV
1
50
MCP(H)
51
LHNGPEEVSHLHLWIGQQSSRDEQ3ACAVLAVHLNTLLGERPVQHREVCG
100
gCap39W
51
LHNGPEEASHLHLWIGQQSSREQGACAVLAVHLNTLLGERPVQHREVQG 100
Mbhl(H)
51
LHNGPEEASHLHLWIGQQSSRDECGACAVLAVHLNTLLGERPVQHRELCG 100
MCP(H)
101 NESDLFHSYFPRGLKYQEGSWHKTSTGA-PAAIKKLYQWGKKNIR 149 II I I I 101 NESDLFHSYFPRGLKYREGGVESWHKTTSGARGAAIRKLYQWGKKNIR 150 IIIIIIIIIII II 101 NESDLFMSYFPRGLKYREGGGRVGISQDNLRATPAAIRKLYQWGKKNIR 150
I
gCap39W Kbhl (H) HCP(H) gCap39(H) Kbhl(H) HCP(H)
150 A T E R A L S W D S F N T G D C F I W L G Q N I F A W C G G K S N I L E R I 51 ATERPLSWDSFNTGDCFILDLGQNIFAWCGGKSNILERNKAPDLALAIR I 151 A T E R A L S W D S F N T G D C F I L D L G Q N I F A W C G G K S N I L E R N K A
199 200 200
200 SERQGKAQVEIVTDGEEPAEH1QVLGPKPALKEGNPEEDLTADKA”NAQ II I 201 SERQGKAQVEIITDGEEPAEHIQVLGPKPALKEGNPEEDITADQTRPNAQ
247
gCap39(H) Kbhl(H1
201 SERQGKAQVEIITDGEEPAEHIQVLGPKPALKEGNPEEDITADQT--NAQ
248
HCP(H)
248 AAALYKVSDATGQKNLTKVADSSPFALELLISDDSFVLDNGLCGKIYIWK I I II 251 AAALYKVSDATGQKNLTKVADSSPFASELLIPDDCFVLDNGLCAQIYIWK
291
gCap39(H) Kbhl(H)
249 AAALYKVSDATGQ~LTKVADSSPFASELLIPDDCFVLDNGLCGKIYIW 298
MCP(H)
298 GRKANEKERQAALQVAEGFISRMQYAPNTQVEILPCGRESPIFKQFFKDWK 348 I I I I 301 GRKANERERQRALQVADGFISWRYSPNTQVEILPCGRESPIFKQFFKNWK 351 I I 299 GMANEKERQAALQVAEGFISFMRYSPNTQVEILRQGRESPIFKQFFKNWK 349
gCap39(M)
401
Endo F
Kbhl(M)
250
300
FIG. 4. Alignment of human MCP to mouse gCap39 and Mbhl. The bars represent mismatched amino acids.
0
10
20
30
Time (min)
FIG. 2. Effects of recombinant MCP on actin filament depolymerization. Pyrene-conjugatedactinfilaments (2 P M stock solution) were diluted 40-fold into varying concentrationsof purified recombinant MCP in 10 mM imidazole buffer, p H 7.5, containing 0.1 M KCI, 1 mM MgCI,, and 0.2 mM CaC12. Fluorescence intensity was monitored over time.
degree of similarity to amino acid sequence foundin theNH2terminal half of gelsolin, sharing a 49% identity with this region but only a 16% identitywith the COOH-terminal half. MCP also shared about the same identity with the NH2terminal (41% identity) and COOH-terminal (10% identity) halves of villin. In addition, MCP demonstratedhomology to the two invertebrate 40-kDa severing proteins, fragmin (30% identity) and severin (25% identity). Human MCP also exhibited 93% identity to the inferred amino acid sequence and an 88% identity to the nucleotide sequence of two mouse cDNAs recently cloned from kidney (32) and fibroblast (33) libraries, respectively named gCap39 and Mbhl (Fig. 4). The inferred amino acid sequence for the human protein contained three substitutions of glutamine for
Molecular Cloning of Human Macrophage Capping Protein
16549
arginine. This change as well as three amino acid deletions, arginine a t amino acid 133, arginine a t amino acid 247, and proline a t amino acid 248 of gCap39, account for the lower ZO! calculated molecular mass of thehumanprotein (38.4 as compared with 39.1 kDa). 111 9 Examination of the MCP sequencefailed to reveal the consensus sequences found in the E F hand structure of the 6t calcium-binding proteins, calmodulin and troponin C(34). 45 Similarly, thismotif has not been found in gelsolin (7). Tissue Distribution of MCP by Western and Northern 29 Blots-Northern blot analysissuggested that MCP expression is restricted. A prominent 1.2-kb band was evident in RNA preparations from human peripheral monocytes and undifferentiated U937 cells (Fig. 5). With the exception of human kidney, which demonstrated a low level of message, MCP mRNA was not detected in other tissues, including skeletal muscle, bladder, liver, brain, and heart. These same blots were hybridized with a“P-labeled human p-actin cDNA probe with specific activity similarto thatof our MCPcDNA probe. After an overnight exposure,all of the RNA preparations revealed the appropriatesize bands for actin mRNA, indicating that the low levels of MCP mRNA were not caused by mRNA degradation (data not shown). Westernblotanalysisusing ahighly specific anti-MCP guinea pig antibody also supporteda restricted tissue andcell I ZS4Sfj78 1 z3430 I O distribution. MCPwas predominantly found inmacrophages. FIG.6. Panels A and R, cellular distribution of MCP by Western T h e protein was not detected in mononuclear cells, polymorwith phonuclear leukocytes, red blood cells, or platelets (Fig. 6, A blot. Cellular extracts were subjected to SDS-PAGE and stained and B). Preparations of spleenextracts revealeda small Coomassie Blue ( A ) or transferred and probed with anti-MCP antibody ( R ) (see “Experimental Procedures”). Lane I, plasma; lane 2, quantity of 41-kDa polypeptide probably derived from splenic red blood cell ghosts; lane 3, platelets; lane 4 , polymorphonuclear macrophages (Fig. 7, A and B). The protein was not detected leukocytes; lane 5 , monocytic cells; lane 6, rabbit macrophage extract. in extracts from rabbit heart, aorta, liver, adrenal, intestine, All extracts were derived from rabbits. 100 pg of extract was loaded bladder, uterus, and skeletal muscle (Fig. 7, A and B ) . Al- in each lane. Molecular mass standards are represented by arrows: though MCP mRNA was present in the kidney, the protein myosin (205 kDa), p-galactosidase (116 kDa), phosphorylase b (9’7.4 kDa), bovine albumin (66 kDa), egg albumin (45 kDa), and carbonic was not demonstrated by Western blots. We also failed to anhydrase (29 kDa). The high molecular mass cross-reactive polypepdetect MCP in plasma. Other investigators claim to have tides in lanes 4 and 5R were also cross-reactivewithsecondary found MCP in mouse plasma (31). It is possible that low antibody alone. This Western blot is representative of four separate levels of MCP may have been released into plasma by dying experiments. Panels C and D,quantitation of MCP concentration in cells.Wehaveusedfreshly prepared plasma separated by rabbit alveolar macrophage extract. C Coomassie Blue-stained and L), nitrocellulose transfers. Lunes 1-4, decreasing concentrations of centrifugation through Ficoll-Hypaque,which may explain rabbit alveolar macrophage extract: 100, 75, 50, and 25 pg, respecthis difference. Experiments were repeated on four separate tively. Lanes 4-8, decreasing concentrations of purified rabbit MCP: occasions. In some instances blots were allowed to develop 1, 0.75, 0.5, and 0.2, respectively. The relative intensity of each until polypeptides cross-reactive with 2” antibody alone be- nitrocellulose band was measured by laser densitometry, generating came apparent. With theexception of mononuclear cells and a linear standard curve andallowing quantification of MCP in macpolymorphonuclear leukocytes in one instance, no 41-kDa rophage extracts. MCP represented between 0.9 and 1% of the total extract protein. Lanes 2-4 demonstrated a linear change in intensity, cross-reactive polypeptides were detected in additionalcell or while lane 1 was saturated, secondary to crowding by adjacent polytissue extracts. peptides. The lower molecular mass band in lanes 5-8 represents a Preliminary surveys of MCP tissue distribution employed degradation product of MCP which is seenwhen the purified protein a low affinity goat anti-MCP antibody. Problems with differ- is stored for prolonged periods. entiating nonspecific background from specific interactions led to the spurious suggestion that MCP might be more widely distributed. Developmentof a much higher affinity and more specific anti-MCP antibody inguinea pigs has allowed a more 18. b b definitive survey of MCP tissue andcell distribution. This high affinity antibody also was used to quantitate the concentration of MCP in rabbit alveolar macrophage cyto1 2 3 4 5 6 7 8 9 FIG.5. Tissue distribution of MCP by Northern analysis. A plasmic extracts. A linear standard curvewas generated using 1.2-kb MCP mRNA is present in undifferentiated U937 cells (lanes varying concentrations of purified rabbit macrophage MCP I and 8 ) analyzed on two separate blots. On the same blot as lane 1 , (see Fig. 6D). The intensityof the 41-kDa reactive band was there are human peripheral monocytes (lane 2 ) , skeletal muscle (lane compared with thereactive polypeptide in varying concentra3 ) , bladder (lane 4 ) , liver (lane 5 ) ,brain (lane 6 ) , and heart (lane 7). tions of macrophage extract (Fig. 6, C and D).These studies On a different blot, MCP mRNA in kidney( l a n e 9) is compared with indicate that MCP represents between 0.9 and 1%of the total U937 cells (lane 8 ) . Positions of 18 S rRNA markers are indicated. protein in rabbit alveolar macrophage cytoplasmic extracts. 10 pg of total RNA was loaded in each lane. The blot was hybridized The lower limit of detection for the anti-MCP antibody in with a full-length ”2P-labeled MCP cDNA (see “Experimental Procedures”). Theexposure times for the autoradiogramswere: overnight Western blots was between 100 and 200 ng of purified MCP (lanes 1-7); 3 days (lanes 8 and 9 ) . (Fig. 6D).
.*
Molecular Cloning
16550
of Human Macrophage Capping
Protein
205-
116. 97.
2 9-
C 1251)ati
i
1 2 3 4 5 6 7
FIG. 7. Tissue distributionof MCP b y W e s t e r nblot analysis. Panel A, Coomassie Blue-stained SDS-PAGE and panel B, nitrocellulose transfers. Lane I , purified rabbit MCP, 1 pg; lane 2, kidney; lane 3, liver; lane 4, spleen; lane 5 , bladder; lane 6, heart; lane 7, skeletal muscle. All tissues were derived from rabbits. 100 pg loaded/ lane. The high molecular mass bands seen inlanes 3 and 4 were also cross-reactive with secondary antibody alone. Molecular mass standards were the same as in Fig. 6. This Western blot is representative of four separate experiments. 00.0
1.o
2.0
3.0
Time (days) Induction of MCP during Cell Differentiation-The results from the tissue distribution all suggest that MCP is mainly FIG. 8. Panel A, Northern blot analysis U937 cells differentiated present in macrophages and macrophage-like cells. In a pre- to macrophages in thepresence of PMA. 10 pg of RNA was extracted liminary experiment we observed that peripheral monocytes at theindicated time points,electrophoresed in a 1.2% formaldehydecultured in complete media with serum, a condition which agarose gel, transferred, and hybridized to MCP and gelsolin cDNA probes. Panel B, the ethidium bromide-stained gel prior to blotting. causes them to acquire macrophage-like properties (35), was The positions of the 18 S and 28 S rRNA markers are indicated by associated with a n increase in MCP mRNA level to greater arrows. Panel C,graphic determination of relative concentrations of than four times that found in monocytes (data not shown). MCP mRNA over time as measuredby laser densitometry of NorthThis finding raised the possibility that MCP mRNA expres- ern blot autoradiograms. Bars represent the standard error of three sion maybe up-regulated as monocyticcellsdevelop into separate experiments. macrophages. T o investigate this possibility in more detail A HRS DAYS the human cell lines U937 and HL60 were induced to differ0 8 1 6 1 2 3 entiate to macrophages by phorbal diester treatment. U937 cells, a monocytic line, and HL60 cells, a promyelocytic line, 285 were both grown in suspension cultures. When induced to differentiate, these cells ceased to proliferate and began to 185 acquire characteristicsof mature macrophages. During differentiation, the cells became noticeably adherent to tissue culas 8 h after PMA treatment.By 1-2 days, ture plastic as early when the majority of the cells wereadhered to the plates, they developed many pseudopodial extensions. The differentiation stages observed were as documentedpreviously for both U937 and HL60 cells (36-38). Total RNA was prepared from thesecells a t different time points after PMA-induced differentiation and analyzed by Northern blots using the 1.2-kb MCP cDNA as a probe. As shown in Fig. 8, undifferentiated U937 cells (time 0),from which the cDNAwascloned, had readily detectable MCP mRNA (relative concentration 2.79 & 0.02 S.E., n = 3, see Time (days) below). Within 16 h of treatment with PMA MCP mRNA FIG. 9. Panel A, Northern blots analysis of HL60 cells differenconcentration began to increase, reaching maximumlevels at tiated to macrophages in the presence of PMA. 10 pg of RNA was 3 days. Quantitation by laser scanning densitometry of the loaded at different time points. Panel B, graphic determination of autoradiograms demonstrated (Fig. 8 B ) that by 16 h message relative concentrations of MCP and mRNAgelsolin (see Fig. 8). had increased to 1.6 times startinglevels (relative concentration 3.80 0.95, n = 3), rising to 2.6 times undifferentiated insignificant change in message over the same time period levels by 3 days (relative concentration 7.33 f 0.13, n = 3). (Fig. 8C, inset). These changeswere compared with gelsolin mRNA. Gelsolin A similar pattern of expression for MCP mRNA was obmessage was barely detectable a t times 0-16 h (relative con- served with PMA-induced differentiation of HL60 cells to centration 0.15-0.19). As compared with MCP, the rise in macrophages (Fig. 9). Levels of MCP mRNA in undifferengelsolin mRNA was delayed, increased levels first being seen tiated cells were lower in thismore primitive cell type (relative at 1 day (relative concentration 1.37 2 0.5, n = 3). The rise concentration 0.68) doubling by 16 h and increasing to greater was more transient, peaking a t 2 days (3.17 & 0.42, n = 3) than 7 times initial levels by 3 days (relative concentration and beginning to decrease at 3 days (2.1 k 0.39, n = 3). 5.30). In HL60 cells, gelsolin message was undetectable prior Analysis of human@-actinmRNA revealed a statistically to differentiation. By 16 h a rise in message was readily
4
*
Molecular Cloning of Human Macrophage Capping Protein detected (relative concentration 1.25), reaching a peak at 2 days (relative concentration 3.71). As observed in U937 cells the levels of actin mRNA remained nearly constant over the time period (relative concentration a t zero time, 4.09; at 3 days, 4.72). The results for gelsolin and actin were similar to previous reports (39). Quantitation of actual protein levels a t various stages of differentiation was not possible because our polyclonal antibody was species specific, reacting only with the rabbit protein but failing to cross-react with human MCP. For our tissue and cell Western blot survey, therefore, only rabbit material was used. Southern Blot Analysis-Southern analysis of human genomic DNA was also performed using the full-length 1.2-kb MCP cDNA. High molecular weight DNA was prepared from three different cell types: U937 cells, human lymphocytes, and a CCL202 human fibroblast line. Restriction digestion with four different enzymes gave essentially the same banding pattern for each of the three DNA samples (Fig. 10). BamHI and Hind111 digestion yielded a single high molecular weight DNA fragment (>23 kb), whereas EcoRI yielded an approximately 15-kb fragmentand SstIdigestion produced two high molecular bands (23 and 7.5 kb). These findings suggest that human MCP is likely to be encoded by a single gene.
16551
strated an apparentbarbed end capping constant which was comparable to that described for rabbit alveolar macrophage capping protein, 0.2-0.4 versus 1 nm. The dissociation constant of the humanprotein for actin filament ends was considerably lower than the mouse recombinant protein (1/ 20-1/40of recombinant gCap39, apparent K,-J 8 nm) (42). This marked difference in actin affinitymay reflect the rapid one-step purification method developed to isolate the human recombinant protein as compared with the more time-consuming three-step method described for recombinant mouse protein purification (42). The higher K Dof the mouse recombinant protein may also reflect the additional amino acids synthesized at the amino-terminal end by their expression system. These differences are less likely to represent aspecies difference since the native mouse protein was found to have a K,-J(1.7 nm) which more closely approximates the human protein (32). Since MCP was originally purified in large quantities from alveolar macrophages, the protein was named macrophage capping protein (17). Northern blot analysis of MCP mRNA by Yu and co-workers (32), however, has suggested this protein may be abundantly expressed in wide variety of tissues whereas analysis by Prendergast and Ziff found the message restricted to thelung and kidney, with smaller amounts being in the heart (33). The present Northern blot survey also DISCUSSION suggests a restricted distribution, MCP mRNA being detected The discovery of MCP cDNA in a human monocyte cell only in U937 cells, peripheral monocytes, and kidney. The line has provided new insights into the relatedness of this differences in Northern blot analysis could be the result of protein to otheractin-severing and barbed end capping pro- monocyte contamination of RNA extracts, differences in hyteins. Analysis of amino acid sequences has revealed that bridization conditions, or duration of autoradiogram exposure. MCP is very similar to the NH2-terminal halves of gelsolin Despite prolonged exposure and increasing RNA loads to 15 pg, in no instance were we able to detect MCP message in and villin as well as to the 40-kDa invertebrateproteins severin and fragmin (40, 41). Unlike the other members of additional tissues. A restricted tissueand cell distribution is also supported by the gelsolin/villin family, MCP does not sever preformed actin filaments. A mouse cDNA was recently discovered with sim- the present Western blot analysis, MCP being primarily deilar molecular weight and function to MCP, called gCap39 by tected in macrophages. This protein was very abundant in one laboratory (31) and h4bhl by a second (32). Comparisons this cell type, representing 0.9-1% of the total cytoplasmic of the derived amino acid sequences of this mouse protein and protein. Aside from splenic tissue which would beexpected to human MCP have revealed a 93% identity, indicating that contain a high concentration of resident macrophages, MCP was not detected in othertissues or cells, indicating that MCP gCap39 and Mbhl aremacrophage capping protein. The calculated molecular mass of the human protein is represented less than 0.2%of thetotal protein in these somewhat lower than themouse protein, 38.4 versus 39.1 kDa samples. On one occasion a cross-reactive 41-kDa polypeptide as a result of the deletion of several basic amino acids. These was detected in polymorphonuclear leukocyte and monocytes, deletions did not impair the human protein’s ability to cap suggesting that these cells may contain levels of MCP near actin filaments. Purified human recombinant MCP demon- the detection limit of our assay. In fibroblasts, MCP was found to represent only 0.01-0.001%of the total cellular protein (32). Unlike gelsolin which has been purified in large amounts from platelets (43, 44), MCP was not detected by Western blotsand could not be purified from platelet extracts. The abundance of MCP in alveolar macrophages as well as this protein’s restricted expression suggest that MCP is likely 23.1 to play an importantrole in macrophage function. This supposition is further supported by Northern blot analysis of 9.4 developing cells. U937 and HL60 cell differentiation to mac6.5 rophage-like cells is associated with a significant rise in MCP 4.3 mRNA levels, 2.6 X in U937 and > 7 x in HL60 cells. As described previously, a rise in gelsolin mRNA is also associated with differentiation. The presence of two barbed end blocking proteins in macrophages may be required to regulate 2.3 actin filament length more tightly during macrophage shape 2.0 change. MCPcan serve to block actin assembly without severing filaments and therefore could more finely control FIG.10. GenomicSouthernblotanalysis of DNA from CCL202 human fibroblast line. 5 pg of DNA was digested with actin filament length during macrophage motile processes. In addition to regulating actin assembly, MCP could serve the indicated enzymes. Samples were then electrophoresed in 0.7% agarose,transferred,andhybridizedwith a full-length 3ZP-MCP other functions in macrophages. Recently MCP was found to cDNA. DNA markers (in kb) are indicated. be concentrated in the nucleus of 3T3 fibroblasts (32). West-
Molecular Cloning
16552
of Human Macrophage Capping Protein
ern blot analysis of nuclear extracts from rabbit alveolar macrophages has also demonstrated the presence of MCP.2 These findings raise the possibility that this protein could control macrophage nuclear structure andfunction. The observations that macrophage capping protein is abundantly expressed only in macrophages and that MCP message rises as monocytes and promyelocytes develop into macrophages indicate that this protein is likely to serve an important andunique functional role in thisphagocytic cell. Determining macrophage capping protein's specific functions in the macrophage is the subject of our ongoing investigations. Acknowledgements-We thank Linda Erwin and Amy Feierstein for their technical assistance and Drs. David Helfman and Daniel Purich for their advice and support. REFERENCES Southwick, F. S., and Stossel, T. P. (1983) Semin. Hematol. 20,305-321 Shalit, M., Dabiri, G. A., and Southwick, F. S. (1987) Blood 70,1921-1927 Howard, T. H., and Meyer, W. H. (1984) J. Cell Biol. 98,1265-1271 Wallace P. J., Westo, R. P., Packman, C. H., and Lichtman, M. A. (1984) J. Celi Biol. 99,1060-1065 5. Carson, M., Weber, A,, and Zigmond, S. H. (1986) J. Cell Biol. 103,27072714 6. Pollard, T. D., and Cooper, J. A. (1986) Annu. Reu. Biochem. 56,987-1035 7. Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten, H. R., and Vin. L . 11986) Nature "", H. ~ ~ . ~. 323.455-457 " ,, 8. AI&, M., Prin auk, E. Finidori, J. Garcia, A., Jeltsch, J. M., Vanderkerchove, J., anflouvard, D. (1988) >Cell Eiol. 107,1759-1766 9. Yin, H. L., Albrecht, J. H., and Fattoum, A. (1981) J. Cell Eml. 9 1 , 9011. 2. 3. 4.
"~
"~ ~~
mr, ""
10. Kwiatkowski, D. J., Mehl, R., Izumo, S., Nadal-Girard, B., and Yin, H. L.
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