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Xenopus laevis Stromal Cell-Derived Factor 1:
Conservation of Structure and Function
During Vertebrate Development
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of May 7, 2017.
Mike Braun, Markus Wunderlin, Kathrin Spieth, Walter
Knöchel, Peter Gierschik and Barbara Moepps
J Immunol 2002; 168:2340-2347; ;
doi: 10.4049/jimmunol.168.5.2340
http://www.jimmunol.org/content/168/5/2340
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Copyright © 2002 by The American Association of
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References
Xenopus laevis Stromal Cell-Derived Factor 1: Conservation of
Structure and Function During Vertebrate Development1
Mike Braun,* Markus Wunderlin,* Kathrin Spieth,* Walter Knöchel,† Peter Gierschik,2* and
Barbara Moepps*
C
hemokines constitute a family of low molecular mass
(8 –11 kDa) and structurally related proteins, with conserved cysteines linked by disulfide bonds (1–3). They
are involved in regulating a wide array of leukocyte functions,
including leukocyte chemotaxis, adhesion, and transendothelial
migration; they modulate angiogenesis and hematopoiesis; and
they block HIV entry into target cells (4 – 8). It has become evident
that chemokines play fundamental roles in development and homeostasis, and function not only in cells of the immune system, but
in many different cell types, including various cells of the CNS (9)
or endothelial cells (10). Four subfamilies, C, CC, CXC, and CX3C
chemokines, are distinguished based on the relative position of
conserved cysteine residues (1, 2).
Stromal cell-derived factor 1 (SDF-1),3 originally identified as a
pre-B cell stimulatory factor (11, 12), is a CXC chemokine produced by many cells and tissues (13–15). The primary structure of
SDF-1 is remarkably conserved across species. Thus, human (h)
Departments of *Pharmacology and Toxicology and †Biochemistry, University of
Ulm, Ulm, Germany
Received for publication August 16, 2001. Accepted for publication December
18, 2001.
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.
1
This work was supported by grants from the Deutsche Forschungsgemeinschaft and
the Medical Faculty of the University of Ulm.
2
Address correspondence and reprint requests to Dr. Peter Gierschik, Department of
Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 11, 89081
Ulm, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: SDF-1, stromal cell-derived factor 1; Sf9 cells,
Spodoptera frugiperda cells; [35S]GTP[S], [35S]guanosine 5⬘-O-(3-thiotriphosphate);
xSDF-1, Xenopus SDF-1; xCXCR4, Xenopus laevis CXCR4; h, human; MALDI-MS,
matrix-assisted laser desorption ionization time-of-flight mass spectrometry; GRO,
growth-related oncogene; cSDF-1, cat SDF-1; mSDF-1, mouse SDF-1.
Copyright © 2002 by The American Association of Immunologists
and feline SDF-1 are identical in amino acid sequence (15, 16).
This high degree of sequence identity between species has previously been taken to suggest that almost all SDF-1 residues are
required for biological activity. Although the genes for other
known hCXC chemokines are located on chromosome 4, the gene
encoding hSDF-1 is present on chromosome 10 (15). Two isoforms, SDF-1␣ (89 aa) and SDF-1␤ (93 aa), differing only by the
presence of four additional amino acids at the very C terminus of
the longer isoform are generated from a single gene by differential
RNA splicing (14, 15). Very recently, a third isoform of SDF-1,
designated SDF-1␥ (119 aa), has been identified in the rat (17).
SDF-1␥ is identical to rat SDF-1␣ in its first 89 residues, but is 30
or 26 residues longer than rat SDF-1␣ and ␤, respectively. The
functional significance of the existence of various mammalian
SDF-1 isoforms is currently unknown. SDF-1 is known to play a
critical role in the regulation of trafficking and transendothelial
migration of leukocytes and in the control of proliferation and
differentiation of several cell types, including hematopoietic and
neural cells (7, 18). Transmembrane signaling of SDF-1 is mediated by CXCR4 (19, 20), a heterotrimeric G protein-coupled chemokine receptor initially identified in leukocytes, and known to
serve as a coreceptor for the entry of T cell-tropic and dual-tropic
HIV into CD4⫹ lymphocytes (8). Although other CXC chemokines can compete for binding to and activation of several distinct
chemokine receptors, SDF-1 is unusual in that it activates a single
receptor, CXCR4. Inactivation of the genes encoding SDF-1 or
CXCR4 in mice caused defects of B lymphopoiesis, myelopoiesis,
gastrointestinal vascularization, and heart ventricular septum formation in the developing embryo (21–24). These observations suggested that the SDF-1/CXCR4 chemokine/receptor system is of
vital developmental importance.
To study the role of chemokines and chemokine receptors as
regulators of early vertebrate development, we set out to isolate
0022-1767/02/$02.00
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Transmembrane signaling of the CXC chemokine stromal cell-derived factor-1 (SDF-1) is mediated by CXCR4, a G proteincoupled receptor initially identified in leukocytes and shown to serve as a coreceptor for the entry of HIV into lymphocytes.
Characterization of SDF-1- and CXCR4-deficient mice has revealed that SDF-1 and CXCR4 are of vital developmental importance. To study the role of the SDF-1/CXCR4-chemokine/receptor system as a regulator of vertebrate development, we isolated
and characterized a cDNA encoding SDF-1 of the lower vertebrate Xenopus laevis (xSDF-1). Recombinant xSDF-1 was produced
in insect cells, purified, and functionally characterized. Although xSDF-1 is only 64 – 66% identical with its mammalian counterparts, it is indistinguishable from human (h)SDF-1␣ in terms of activating both X. laevis CXCR4 and hCXCR4. Thus, both xSDF-1
and hSDF-1␣ promoted CXCR4-mediated activation of heterotrimeric Gi2 in a cell-free system and induced release of intracellular
calcium ions in and chemotaxis of intact lymphoblastic cells. Analysis of the time course of xSDF-1 mRNA expression during
Xenopus embryogenesis revealed a tightly coordinated regulation of xSDF-1 and X. laevis CXCR4. xSDF-1 mRNA was specifically
detected in the developing CNS, incipient sensory organs, and the embryonic heart. In Xenopus, CXCR4 mRNA appears to be
absent from the heart anlage, but present in neural crest cells. This observation suggests that xSDF-1 expressed in the heart anlage
may attract cardiac neural crest cells expressing CXCR4 to migrate to the primordial heart to regulate both septation of the
cardiac outflow tract and differentiation of the myocardium during early heart development. The Journal of Immunology, 2002,
168: 2340 –2347.
The Journal of Immunology
Materials and Methods
Materials
The production of baculoviruses encoding xCXCR4, hCXCR4, rat G protein ␣i2 subunit, and both human G protein ␤1 subunit and bovine G protein
␥3 subunit is described in Ref. 25. The N33A analog of hSDF-1␣ (27) was
prepared by chemical synthesis, and was generously provided by Dr. M. A.
Siani (Gryphon Sciences, South San Francisco, CA). [35S]Guanosine 5⬘O-(3-thiotriphosphate) ([35S]GTP[S]) was obtained from NEN (Boston,
MA). X. laevis embryos were prepared and staged according to Refs. 28
and 29. A X. laevis spleen cDNA library was obtained from the Resource Center/Primary Database within the German Human Genome
Project at the Max-Planck Institute for Molecular Genetics (Berlin, Germany; http://www.rzpd.de).
Screening of a X. laevis cDNA library
The X. laevis spleen cDNA library was probed with a 32P-labeled mouse
SDF-1␣ cDNA fragment (nt 145–351, European Molecular Biology Laboratory (EMBL)/GenBank Data Libraries accession no. L12029). Positive
clones were analyzed by restriction endonuclease mapping and DNA
sequencing.
Production of recombinant baculoviruses
The xSDF-1 cDNA was digested with HindIII and EcoRI. The resulting
fragment was filled in using Klenow enzyme and subcloned into the SmaI
site of the baculovirus transfer vector pVL1393 (Invitrogen, San Diego,
CA). The correct orientation of the insert was verified by DNA sequencing.
Recombinant baculoviruses were obtained by transfecting Spodoptera
frungiperda cells (Sf9 cells; Invitrogen) with a 25/1 mixture of the transfer
vector and a modified baculovirus DNA (BaculoGold, BD PharMingen,
San Diego, CA), which contains a lethal deletion and is rescued by the
DNA of the transfer vector. High-titer stocks of the baculoviruses were
obtained by two cycles of amplification in Sf9 cells.
Production of recombinant xSDF-1
For production of recombinant xSDF-1, Trichoplusia ni 5B1-4 cells (High
five cells; Invitrogen) were grown at 27°C in suspension culture in InsectXPRESS medium (BioWhittaker, Walkersville, MD) supplemented with
0.2% (v/v) Pluronic F-68 (Life Technologies, Grand Island, NY), 0.5
mg/ml gentamicin (Life Technologies), and 2.5 ␮g/ml amphotericin B
(Fungizone; Life Technologies) in 1800 ml Fernbach culture flasks. Cells
(8 ⫻ 108/flask) were incubated for 48 h with recombinant baculovirus in
400 ml medium/flask at 80 rpm on a rotary shaker with an amplitude of 25
mm. The medium containing recombinant xSDF-1 was collected from the
cell suspension by centrifugation at 80,000 ⫻ g for 30 min at 4°C. The
supernatant was passed through 0.22-␮m pore size nitrocellulose filters,
snap-frozen in liquid nitrogen, and stored at ⫺80°C.
Purification of recombinant xSDF-1
Recombinant xSDF-1 was purified from the culture supernatant by sequential chromatography on Heparin Sepharose High Performance and
SOURCE 15S using a Pharmacia ÄKTAexplorer chromatography system
(Amersham Pharmacia Biotech, Piscataway, NJ). The filtered supernatant
(150 ml, 230 mg of protein) was applied to a 5 ml HiTrap Heparin column
(Amersham Pharmacia Biotech) that had been equilibrated with buffer A
(10 mM Na2HPO4/NaH2PO4, pH 7.3). The flow rate was 3.5 ml/min. After
application of the sample, the column was washed with 60 ml of buffer A
and eluted with a linear gradient (50 ml) of NaCl (0 –2 M) in buffer A.
Fractions of 2 ml were collected and analyzed by SDS-PAGE and CXCR4mediated [35S]GTP[S]-binding. The active material, which eluted at ⬃1.1–
1.5 M NaCl, was pooled (12 ml, 3.6 mg of protein) and diluted with 10 mM
Na2HPO4/NaH2PO4, pH 7.3, to obtain a final NaCl concentration of 0.1 M.
The sample was then applied to a 1 ml RESOURCE S column (Amersham
Pharmacia Biotech), which had been equilibrated with buffer A. The flow
rate was 2 ml/min. After application of the sample, the column was washed
with 15 ml of buffer A and eluted with a linear gradient (40 ml) of NaCl
(0 –1 M) in buffer A. Fractions of 1 ml were collected and analyzed by
SDS-PAGE and CXCR4-mediated [35S]GTP[S]-binding. The active material eluted at ⬃0.5– 0.6 M NaCl.
Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-MS)
Molecular mass determination of proteins was performed by delayed extraction MALDI-MS on a Bruker REFLEX III time-of-flight spectrometer
(Bruker-Daltonics, Bremen, Germany) equipped with a UV nitrogen laser
(337 nm). The samples were concentrated and desalted using ZipTip C18
pipette tips (0.6 ␮ l resin) as recommended by the manufacturer (Millipore,
Bedford, MA). The samples were eluted from the C18 matrix in acetonitrile/0.1% (v/v) trifluoroacetic acid, 2/1 (v/v). One microliter of this solution was mixed with 1 ␮l of saturated ␣-cyano-4-hydroxycinnamic acid
solution in acetonitrile/0.1% (v/v) trifluoroacetic acid, 2/1 (v/v). Spectra
were recorded after evaporation of the solvent. The singly and doubly
charged ion signals from bovine ubiquitin (Mr ⫽ 8565.8510) were used for
external mass calibration of all mass spectra.
Membrane preparation of baculovirus-infected insect cells
Sf9 cells were grown at 27°C in 59 cm2 cell-culture dishes in TNM-FH
medium (T 1032; Sigma Aldrich, St. Louis, MO) supplemented with 10%
FCS and 0.5 mg/ml gentamicin. For production of recombinant receptors
and heterotrimeric Gi2, cells were grown to a density of ⬃60%, and incubated for 1 h at 27°C in 2 ml per dish of medium containing the recombinant baculovirus(es). The cells were then supplemented with 9 ml per
dish of fresh medium and maintained in this medium for 48 h at 27°C.
Infected cells were suspended in medium, pelleted by centrifugation, and
resuspended in 600 ␮l per dish of ice-cold lysis buffer containing 20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 3 ␮M GDP, 2 ␮g/ml soybean trypsin
inhibitor, 1 ␮M pepstatin, 1 ␮M leupeptin, 100 ␮M PMSF, and 1 ␮g/ml
aprotinin. Cells were homogenized by forcing the suspension six times
through a 0.5 ⫻ 23 mm needle attached to a disposable syringe. After 30
min on ice, the lysate was centrifuged at 2,450 ⫻ g for 45 s to remove
unbroken cells and nuclei. A crude membrane fraction was isolated from
the resulting supernatant by centrifugation at 26,000 ⫻ g for 30 min at 4°C.
The pellet was rinsed with 300 ␮l of lysis buffer, resuspended in 300 ␮l of
fresh lysis buffer, snap-frozen in liquid nitrogen, and stored at ⫺80°C.
[35S]GTP[S] binding
Binding of [35S]GTP[S] to membranes of baculovirus-infected insect cells
was assayed as described (30). In brief, membranes (10 ␮g of protein per
sample) were incubated for 60 min at 30°C in a mixture (100 ␮l) containing 62.5 mM triethanolamine/HCl, pH 7.4, 1.25 mM EDTA, 6.25 mM
MgCl2, 95 mM NaCl, 3.75 ␮M GDP, and 0.34 nM [35S]GTP[S] (1,250
Ci/mmol). The incubation was terminated by rapid filtration through
0.45-␮m pore size nitrocellulose membranes (Advanced Microdevices,
Ambala Cantonment, India). The membranes were washed and dried, and
the retained radioactivity was determined by liquid-scintillation counting.
Nonspecific binding was defined as the binding not competed for by 60 ␮M
unlabeled GTP[S].
Measurement of cytosolic free Ca2⫹ concentration
CCRF-CEM cells (2 ⫻ 106 cells) were loaded with fura 2 by incubation for
30 min at 37°C in 1 ml of Ca2⫹ flux buffer (20 mM HEPES/NaOH, pH 7.4,
1 mM CaCl2, 4.6 mM KCl, and 136 mM NaCl) containing 1 nM of the
acetoxymethyl ester of fura 2 (Molecular Probes, Eugene, OR). The cells
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and characterize their cDNAs from Xenopus laevis, an organism
widely used to study vertebrate embryogenesis. Recently, we isolated and characterized a cDNA encoding CXCR4 of X. laevis
(xCXCR4; Ref. 25). xCXCR4 mRNA expression was up-regulated
during early neurula stages and remained high during early organogenesis. Whole-mount in situ hybridization analysis showed
abundant expression of xCXCR4 mRNA in the nervous system,
including forebrain, hindbrain, and sensory organs, and in neural
crest cells. To identify the ligand which activates xCXCR4 in Xenopus, we have now isolated a cDNA encoding Xenopus SDF-1
(xSDF-1). The encoded polypeptide, designated xSDF-1, was
functionally characterized, and the expression of its mRNA was
determined during embryonic development and in the adult frog.
The results revealed that xSDF-1 differs considerably in primary
structure from its mammalian counterparts, but is nevertheless undistinguishable from hSDF-1 in terms of activating both hCXCR4
and xCXCR4. Our findings not only allow identification of key
residues of SDF-1 and CXCR4 involved in agonist binding and
receptor activation, but also show that these residues have been
maintained over a period of at least 325 million years, which is the
approximate evolutionary distance between Xenopus and living
mammals (26). The latter is in support of a pivotal role of this
chemokine receptor pair in vertebrate development.
2341
2342
were washed and resuspended in the same buffer (1 ml), and 5 ␮l of
xSDF-1 in 10 mM Na2HPO4/NaH2PO4, pH 7.3 was added. Fura 2 fluorescence was measured on a fluorescence spectrophotometer (LS50B,
PerkinElmer, Wellesley, MA) thermostated at 37°C. Excitation and emission wavelengths were 340/380 and 510 nm, respectively.
Chemotaxis assay
The migration of human acute lymphoblastic leukemia cells (CCRF-CEM,
American Type Culture Collection (Manassas, VA) certified cell line 119)
was assessed in disposable Transwell trays (Costar, Cambridge, MA) with
6.5-mm diameter chambers and 3-␮m pore-size polycarbonate membranes.
In brief, chemokines were diluted in HEPES-buffered RPMI 1640 (Life
Technologies) supplemented with 10 mg/ml BSA and added to the lower
compartments, and CCRF-CEM cells (106 cells) were added in the same
medium (100 ␮l) to the upper compartments. After an incubation for 2 h
at 37°C in a humidified CO2 atmosphere (10%), the filters were removed
from the chamber, washed in PBS, fixed, and stained with Diff-Quik (Dade
Behring, Marburg, Germany) according to the manufacturer’s instructions.
The number of cells migrated through the membrane was determined by
microscopical examination in three randomly selected fields of three independent Transwell chambers at ⫻40 magnification.
RT-PCR analysis of xSDF-1 mRNA expression
Whole-mount in situ hybridization of X. laevis embryos
The localization of xSDF-1 transcripts in Xenopus embryos was analyzed
by using the whole-mount in situ hybridization technique (31) with some
modifications (28). In brief, embryos were fixed for 90 min at room temperature in freshly prepared MEMPFA(0.1 M MOPS, pH 7.4, 2 mM
EGTA, 1 mM MgSO4, and 4% (w/v) paraformaldehyde), and then stored
at ⫺20°C in ethanol. Whole-mount in situ hybridization was performed
with digoxigenin-labeled antisense cRNA transcribed in vitro using T7
RNA polymerase from a xSDF-1 cDNA fragment (nt 1–341) using the
digoxigenin RNA Labeling kit (SP6/T7; Boehringer Mannheim, Indianapolis, IN). Labeled antisense RNA transcripts were localized by alkaline
phosphatase-conjugated anti-digoxigenin Abs and Boehringer Mannheim
purple substrate (Boehringer Mannheim). After final fixation of the embryos in MEMPFA for at least 2 h, they were bleached in 30% H2O2/
methanol (33/67, by volume; 10% H2O2 final), dehydrated in pure methanol, and stored at 4°C in pure methanol. To enhance the transparency of
the embryos, they were incubated for 60 min at 20°C in benzylbenzoate/
benzyl alcohol (2/1, by volume).
Miscellaneous
Radiolabeled cDNA probes were prepared by priming with random hexanucleotides (32). Blots were hybridized with radiolabeled probes as descibed in Ref. 33, except that the final wash was done at 64°C in a solution
of 2⫻ SSC and 0.1% (w/v) SDS. DNA was sequenced on an ABI Prism
310 Genetic Analyzer using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA Polymerase, FS (Applied Biosystems, Foster City, CA). Protein concentrations were determined according to Bradford (34) using bovine IgG as standard.
Results
Isolation of a SDF-1 cDNA from a X. laevis cDNA library
To determine the primary structure of the X. laevis homolog of
mammalian SDF-1, a X. laevis spleen cDNA library was screened
at low stringency using a radiolabeled cDNA fragment of mouse
SDF-1␣ as a probe. DNA sequence analysis and comparison of the
predicted amino acid sequences of three independent clones to the
sequences of known chemokines revealed that all three cDNAs
encoded a protein showing high sequence similarity to mammalian
SDF-1. One of these cDNAs (Resource Center/Primary Database
clone ID: HACHp412I1133Q2) was sequenced by primer walking.
The cDNA encoded a polypeptide of 94 aa with a calculated Mr of
10,701, which was tentatively designated as xSDF-1. The 5⬘ noncoding region of this cDNA started at position ⫺31 from the ATG
initiation codon. No other in-frame ATG codon was present in the
5⬘ noncoding region. The 3⬘ noncoding region consisted of ⬃1.9
kb. Fig. 1 shows an alignment of the amino acid sequences of
xSDF-1 and SDF-1␤ of man (hSDF-1␤), mouse (mSDF-1␤), and
FIGURE 1. Comparison of the amino acid sequences of X. laevis and mammalian SDF-1. The amino acid sequences of xSDF-1, hSDF-1␤, mSDF-1␤,
cSDF-1␤, and mouse KC were aligned using the program CLUSTAL W (53) contained in the OMIGA 2.0 software package (Oxford Molecular, Oxford,
U. K.). Residues present in all SDF-1 polypeptides, but absent from KC, are shown in red, those present in both SDF-1 and KC are shown in gray. The
amino acid sequence numbering starts with the lysine residue present at the amino terminus of mature SDF-1 (12). The positions of the structural elements
of hSDF-1 (41) are indicated. The nucleotide sequence of xSDF-1 has been deposited with the EMBL/GenBank Data Libraries under accession no.
AJ278857. The amino acid sequences of hSDF-1␤, mSDF-1␤, cSDF-1␤, and KC correspond to SWISSPROT databank entries P48061, P40224, O62657,
and P12850, respectively.
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Total RNA was prepared from X. laevis embryos and adult tissues using
TRIzol Reagent as recommended by the supplier (Life Technologies). Oligo(dT)-primed sscDNA was made from total RNA after DNase I digestion
(amplification grade; Life Technologies) using the SuperScript preamplification system (Life Technologies). The amounts of sscDNA used as templates for the amplification of the xSDF-1 cDNAs were adjusted to similar
levels according to the amount of single-stranded X. laevis histone H4
cDNA present in the sample, as determined by semiquantitative PCR. The
xSDF-1 cDNA was amplified from these samples by PCR (28 cycles: 94°C
for 30 s, 59°C for 45 s, 72°C for 45 s, followed by a single incubation at
72°C for 10 min) using primers P1, 5⬘-CACAGCTCCAGCCACAA
CATG-3⬘ (nt 14 –34, sense), and P2, 5⬘-GCCAGAACACTAACAAA
GAAATTA-3⬘ (nt 315–338, antisense). The numbering of all oligonucleotides used as primers in this study refers to the nucleotide sequence of
xSDF-1 cDNA deposited in the EMBL/GenBank Data Libraries under accession no. AJ278857. The PCR products contained in 8 ␮l of the reaction
volume were fractionated by agarose (1% (w/v)) gel electrophoresis, transferred to a nylon membrane (Biodyne B; Pall, Dreieich, Germany), and
sequentially hybridized with radiolabeled xSDF-1 (nt 1–386) and Xenopus
histone H4 cDNA probes (nt 1491–1679, EMBL/GenBank accession no.
X03017).
X. laevis STROMAL CELL-DERIVED FACTOR 1
The Journal of Immunology
2343
Production of the recombinant xSDF-1
To obtain mature xSDF-1 as a recombinant protein, the cDNA was
cloned into the baculovirus transfer vector pVL1393 and a recombinant baculovirus was produced. Insect cells were infected with
the virus, and recombinant mature xSDF-1 was purified from the
culture medium by sequential heparin affinity and cation exchange
chromatography. Analysis of the fractions eluting from the cation
exchange resin by SDS-PAGE revealed that a mostly homogeneous protein of the expected molecular mass was obtained at
⬃0.5– 0.6 M NaCl (Fig. 2A). The purified protein was subjected to
molecular mass determination by MALDI-MS (Fig. 2B). The measured molecular mass was 8,469.03 Da, which is in excellent
agreement with the calculated mass ([M ⫹ H]⫹) of mature xSDF-1
of 73 aa (K1-T73, cf Fig. 1) containing four half-cystines
(8,469.08 Da). In addition, two peptides with smaller measured
molecular masses (8,243.79 and 8,057.52) were present in the
same peak. These peptides most likely correspond to the V3-T73
and L5-T73 forms of xSDF-1. The yield of purified protein was
typically ⬃1 ␮g/ml of starting culture supernatant.
FIGURE 2. Purification on RESOURCE S and mass spectrometric
analysis of recombinant xSDF-1. A, Recombinant xSDF-1 was partially
purified from the culture supernatant of High five insect cells infected with
baculovirus encoding xSDF-1 by sequential chromatography on Heparin
Sepharose High Performance and SOURCE 15S. Fractions containing
xSDF-1 were pooled and subjected to chromatography on RESOURCE S.
Aliquots of the indicated fractions were analyzed by SDS-PAGE. Proteins
were stained with Coomassie blue (inset). The positions of the molecular
mass standards (bovine ␣-lactalbumine, 14.4 kDa; bovine aprotinin, 6.5
kDa) are indicated. B, Fraction 24 was analyzed by MALDI-MS. An expansion of the [M ⫹ H]⫹ molecular ion region of the spectrum is shown.
The corresponding peptides are indicated. Assuming that the relative
amounts of the three nearly identical components can be deduced by comparing the areas under the curve of the corresponding peaks (54), we estimate that the L5-T73, V3-T73, and K1-T73 forms of xSDF-1 are present
in this preparation at 4, 67, and 29% (w/w), respectively.
Functional reconstitution of xCXCR4 and hCXCR4 with
heterotrimeric Gi2
vates not only Xenopus, but also hCXCR4 and thus, confirm that
xSDF-1 is the amphibian counterpart of mammalian SDF-1. Furthermore, the results introduce xSDF-1 as the first functional nonmammalian chemokine to be described.
To investigate whether xSDF-1 does in fact represent an agonist of
CXCR4, including xCXCR4, rCXCR4 of Xenopus (xCXCR4) and
man (hCXCR4) were reconstituted with recombinant heterotrimeric Gi2 by coinfection of Sf9 insect cells with baculovirus encoding either xCXCR4 or hCXCR4, and a combination of baculoviruses encoding the Gi2 subunits ␣i2 and ␤1␥3. Receptor-G
protein interaction was assayed by measuring the effect of
hSDF-1␣ and xSDF-1 on the binding of [35S]GTP[S] to insect cell
membranes. Fig. 3 shows that xSDF-1 (200 nM) and hSDF-1␣
(200 nM) caused similar (⬃4-fold) activation of xCXCR4 and
hCXCR4. Both xSDF-1 and hSDF-1␣ failed to increase
[35S]GTP[S]-binding in membranes expressing ␣i2䡠␤1␥3 in the absence of xCXCR4 or hCXCR4. No activation of [35S]GTP[S]binding was observed upon testing xSDF-1 at the same concentration (200 nM) on membranes of insect cells coexpressing other
CXCRs, e.g., CXCR1 and 2, and ␣i2䡠␤1␥3 (data not shown). Taken
together, these results demonstrate that xSDF-1 specifically acti-
Release of intracellular Ca2⫹ and chemotaxis
There is evidence to suggest that chemically distinct agonists may
produce arrays of active states of a single G protein-coupled receptor to differentially activate the G proteins of a given cell, and
thus, produce distinct cellular responses (37– 40). Therefore, the
effects of xSDF-1 and hSDF-1␣ on hCXCR4-mediated responses
of intact human cells were examined and compared. Fig. 4 shows
that xSDF-1 induced a rapid and transient rise in the concentration
of cytoplasmic free calcium, intracellular Ca2⫹ concentration, in
human acute lymphoblastic leukemia (CCRF-CEM) cells. The intracellular Ca2⫹ concentration increase was very similar both in
terms of its kinetics and its magnitude to the increase observed
upon testing hSDF-1␣ at the same concentration. xSDF-1 and
hSDF-1␣ also caused very similar concentration-dependent chemotactic responses of CCRF-CEM cells with maximal effects at
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cat (cSDF-1␤) as well as KC, the mouse equivalent of human
growth-related oncogene (GRO)-␣, GRO-␤, and/or GRO-␥ (19,
35). KC was the closest relative of xSDF outside the SDF-1 family
identified in the National Center for Biotechnology Information
databases using the basic local alignment search tool algorithm.
xSDF shares 64 – 66 and 26% identical amino acids with the mammalian SDF-1 polypeptides and KC, respectively. Thus, the degrees of amino acid identity between xSDF-1 and its mammalian
counterparts (64 – 66%) are strikingly lower than the degrees obtained when mammalian SDF-1 polypeptides are compared with
each other (92–96%). Analysis of the xSDF-1 sequence using a
signal-sequence-detecting algorithm (36) led to the prediction of
three potential signal sequence cleavage sites with similar scores
between residues Gly21 and Lys22, Cys17 and Leu18, Thr19 and
Glu20 (scores: 8.57, 6.71, and 6.57, respectively). Assuming that
the former site is the major cleavage site in Xenopus, as it is in the
mouse (12), the mature xSDF-1 polypeptide consists of 73 aa with
a calculated Mr of 8,468. Although the mature hSDF-1␤ and
cSDF-1␤ proteins are identical and differ from the mature
mSDF-1␤ by only two residues, mature xSDF-1 differs by 21 residues from its mammalian counterparts (white in Fig. 1). Eighteen
of the remaining 52 identical residues are also found in the nonSDF-1 CXC chemokine KC (pink in Fig. 1), 34 residues are
uniquely present in SDF-1 (red in Fig. 1).
2344
FIGURE 3. Activation of xCXCR4 and hCXCR4 by hSDF-1␣ and
xSDF-1. Sf9 cells were coinfected with baculoviruses encoding either
xCXCR4 or hCXCR4, and the G protein subunits ␣i2 and ␤1␥3. Cells were
homogenized and fractionated into soluble and particulate fractions, and
aliquots of the particulate fractions (10 ␮g protein/sample) were incubated
with 0.34 nM [35S]GTP[S] in the absence or presence of 200 nM SDF-1␣
or xSDF-1. The samples were analyzed for bound [35S]GTP[S] by rapid
filtration and scintillation counting. Each value represents the mean ⫾ SD
of triplicate determinations.
FIGURE 5. Stimulation of CCRF-CEM cell chemotaxis by xSDF-1 and
hSDF-1␣. The effect of xSDF-1 and hSDF-1␣ on chemotaxis of human
acute lymphoblastic leukemia (CCRF-CEM) cells was assessed in disposable Transwell trays with 6.5-mm diameter chambers equipped with 3-␮m
pore size polycarbonate membranes. The chemotactic response to xSDF-1
is plotted as a function of the concentrations of the full-length (K1-T73),
active constituent of recombinant xSDF-1 and of full-length (K1-K68) recombinant hSDF-1␣. The values shown represent the mean ⫾ SD of the
cell numbers determined in three independent Transwell chambers.
Expression of xSDF-1 mRNA in adult X. laevis tissues and in
X. laevis embryos
The expression of xSDF-1 mRNA in tissues of adult X. laevis was
assessed by RT-PCR analysis (Fig. 6). Using this methodology,
high levels of xSDF-1 mRNA were detected in spleen, kidney,
lung, stomach, testis, and skeletal muscle. Lower levels were observed in liver and heart. Few transcripts were found present in
brain and skin. No xSDF-1 mRNA was detected in the ovary. The
temporal and spatial pattern of xSDF-1 mRNA expression during
embryogenesis was assessed by RT-PCR analysis and wholemount in situ hybridization (Figs. 7 and 8). RT-PCR analysis of
RNA prepared from X. laevis embryos at different stages of embryonic development revealed a sharp rise of xCXCR4 mRNA
expression between stages 14 and 18 to a level increasing further
until stage 45 (Fig. 7). Results obtained by whole-mount in situ
hybridization analysis were consistent with this pattern. As shown
in Fig. 8A, xSDF-1 mRNA expression was up-regulated during
organogenesis (stages 21–23) in the anterior part of the embryo,
where the regional segregation of the neural tube into fore-, mid-,
and hindbrain takes place (29). In stage 32–34 embryos, xSDF-1
mRNA was detected in the mid- and hindbrain, otic vesicles and
FIGURE 4. Effects of xSDF-1 and hSDF-1␣ on the concentration of
cytosolic free Ca2⫹ in intact CCRF-CEM cells. Human acute lymphoblastic leukemia (CCRF-CEM) cells were loaded with fura 2. At the times
indicated by arrows, xSDF-1 (20 nM) or hSDF-1␣ (20 nM) were added.
The fluorescence due to intracellular Ca2⫹ was recorded.
eyes, and the dorsal fin (Fig. 8B). Furthermore, xSDF-1 transcripts
were present within the posterior heart anlage, where the pericardial mesoderm begins to form the dorsal mesocardium at stage 32
(29). At stages 39 – 40, xSDF-1 mRNA was found highly expressed in the proctodeum located in the posterior part of the embryo (Fig. 8A).
Discussion
In this study, we have isolated and characterized a cDNA encoding
a X. laevis homolog of mammalian SDF-1. The encoded protein
consists of 94 aa and is only 64 – 66% identical to its mammalian
counterparts. The degree of amino acid identity is particularly low
within the predicted signal sequence, Met⫺20-Gly⫺1, where only 4
of 21 residues (excluding the initiating methionine) are shared between Xenopus and mammalian SDF-1. The three-dimensional
structure of hSDF-1 is made up of three antiparallel ␤-strands (␤1,
residues 24 –31; ␤2, residues 35– 42; ␤3, residues 45– 49; cf Fig. 1)
followed by a C-terminal ␣-helix (residues 56 – 64) that is packed
FIGURE 6. RT-PCR analysis of xSDF-1 mRNA expression in tissues
of adult X. laevis. Total RNA was prepared from adult X. laevis spleen,
kidney, lung, liver, heart, brain, stomach, testis, ovary, muscle, and skin
and used as template for RT-PCR. The amounts of sscDNA used as templates for the amplification of the xSDF-1 cDNA (upper panel) were adjusted to similar levels according to the amount of single-stranded X. laevis
histone (H4) cDNA present in the sample (lower panel), as determined by
semiquantitative PCR. The amplified DNA fragments were fractionated by
agarose (1% (w/v)) gel electrophoresis, transferred to a nylon membrane,
and hybridized with radiolabeled xSDF-1 (upper panel) or X. laevis histone
H4 (lower panel) cDNA fragments. A sample containing no singlestranded template cDNA was analyzed for comparison (Control).
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⬃10 nM SDF-1 (Fig. 5). Thus, xSDF-1 and hSDF-1␣ are indistinguishable in regulating complex functions of intact cells by activating a single species of CXCR4.
X. laevis STROMAL CELL-DERIVED FACTOR 1
The Journal of Immunology
FIGURE 7. RT-PCR analysis of xSDF-1 mRNA expression in X. laevis
embryos. Total RNA was prepared from from X. laevis embryos (stages
2–34; Ref. 29), and used as template for RT-PCR. The amounts of sscDNA
used as templates for the amplification of the xSDF-1 cDNA (upper panel)
were adjusted to similar levels according to the amount of single-stranded
X. laevis histone (H4) cDNA present in the sample (lower panel), as determined by semiquantitative PCR. The amplified DNA fragments were
fractionated by agarose (1% (w/v)) gel electrophoresis, transferred to a
nylon membrane, and hybridized with radiolabeled xSDF-1 (upper panel)
or X. laevis histone H4 (lower panel) cDNA fragments. A control sample
containing no single-stranded template cDNA was analyzed for comparison (C).
FIGURE 8. Whole-embryo in situ analysis of xSDF-1 mRNA expression during X. laevis embryogenesis. A, Digoxigenin-labeled xSDF-1 antisense RNA was prepared and hybridized to Xenopus embryos (stages
21–23, 32–34, and 39 – 40). For enhancement of transparency, the embryos
were treated with benzylbenzoate/benzyl alcohol. B, Stage 32–34 embryo
(e, eye; b, brain; ov, otic vesicle; and h, heart anlage).
(100%), followed by the amino terminus, ␤1, and ␤2 (each 88%),
the extended loop (57%), ␤3 (40%), and the 310 helix (25%;
Fig. 1).
The high degree of sequence identity within the region corresponding to the flexible amino-terminus of hSDF-1 is consistent
with the observation that this region is directly involved in highaffinity binding of SDF-1 to and activation of hCXCR4 (12, 42).
The fact that a hydrophobic valine residue is present in position 6
of xSDF-1 in place of the polar serine present in mammalian
SDF-1 implies that this residue is not involved with the agonistreceptor interaction. Crump et al. (42) have recently proposed a
two-site model for the interaction of SDF-1 with mammalian
CXCR4 (43). According to this model, binding of SDF-1 is initiated by a direct interaction (“docking step”) between a sequence
motif immediately adjacent to the second cysteine residue,
R12FFESH (site 1), with the amino-terminal segment of CXCR4.
The docking permits access of the flexible amino terminus of
SDF-1 (site 2) to a more buried receptor site. The second binding
step induces a conformational change of the receptor transmembrane helices that allows intracellular G protein activation. Very
recently, the major conformation of the amino-terminal 17 residues
of mature mammalian SDF-1 (K1PVSLSYRCPCRFFESH) was
determined by nuclear magnetic resonance, and found to consist of
two similar ␤-turns of the ␤-␣R type made up of residues 5– 8 and
11–14 (44). The authors noticed that the CRF portion of the second
␤-turn is a partial palindrome of the tail of the 9-mer sequence
(K1PVSLSYRC) involved in the formation of the first ␤-turn. This
might explain the enhanced affinity of a 9-mer dimer (residues 1–9
linked by a disulfide bond at residue 9) relative to monomeric
9-mer to activate the receptor (45). The fact that this palindome is
perfect in xSDF-1 is in support of this notion. In any case, two of
six residues of the “RFFESH motif,” corresponding to Phe13 and
His17 of mammalian SDF-1, appear to be dispensable for the docking process, since these residues are replaced by tyrosine and asparagine, respectively, in xSDF-1. In this context, the RFFESH
motif might be more appropriately referred to as R(Y/F)FES motif.
The high degree of sequence identity between Xenopus and
mammalian SDF-1 in regions downstream of the R(Y/F)FES motif
is surprising in light of previous observations demonstrating that
the first 17 residues of hSDF-1 are sufficient for binding to and
activation of CXCR4 (42, 45, 46). Compared with the low degree
of sequence conservation within the presumed signal peptide, the
conservation is particularly striking in the regions corresponding to
the ␤1 and ␤2 strands and to the C-terminal ␣-helix, all of which
contain a high proportion of the residues present in both Xenopus
and mammalian SDF-1, but absent from the closest SDF-1 relative, KC (red in Fig. 1). The high degree of conservative evolutionary pressure to maintain these residues over the past 325 million years, which is the approximate evolutionary distance between
Xenopus and living mammals (26), strongly suggests that these
residues make up functionally important parts of the SDF-1 molecule. Although it seems possible that some of the conserved residues are involved in maintaining the overall tertiary structure, and
possibly the stability of SDF-1, it is important to note that a chimeric CXC chemokine, designated GROH2, sharing with SDF-1
only 6 of the 24 residues specifically present in SDF-1 (red in Fig.
1) downstream of the R(Y/F)FES motif (His25, Ile28, Thr31, Asn33,
Glu63, Asn67) bound to and activated CXCR4 with Kd and EC30
values corresponding closely to the values of native SDF-1␣ and
SDF-1␤ (42). Thus, the conserved residues may be involved in
serving functions of SDF-1 other than interaction with CXCR4 and
maintenance of protein structure and/or stability. For example,
structure function analysis of human SDF-1␣ has shown that three
␤1-strand residues of hSDF-1␣, Lys24, His25, and Lys27 either
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against the ␤-sheet (41, 42). The first ␤-sheet is preceeded by a
disordered amino-terminal region (residues 1– 8), an extended loop
(residues 12–18), and a single turn of a 310 helix (residues 19 –22;
Refs. 41 and 42). An alignment of the primary structures of Xenopus and mammalian SDF-1 with these structural elements revealed that the degree of sequence identity between the mature
SDF-1 polypeptides is particularly high for the C-terminal ␣-helix
2345
2346
mal differentiation and function of the myocardium during early
heart development (52).
Acknowledgments
We thank Stephan Rütten, Ingrid Büsselmann, Karin Dillinger, and
Susanne Gierschik for expert technical assistance.
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