(Received for publication, August 30, 1996, and in revised form, January 8, 1997)
From Recently, we identified a novel putative human
cytokine expressed by activated CD8+ T cells, which
was designated ATAC (ctivation-induced,
cell-derived, nd hemokine-related; the same
molecule has been identified independently as lymphotactin and single
cysteine motif-1). In this report, we provide evidence that ATAC is a
secreted 93-amino acid protein that is generated from its precursor by
proteolytic cleavage between Gly21 and Val22.
An estimated 60% of ATAC (Val22-Gly114) is
secreted as an unmodified protein with a molecular mass of 10,271.72 Da
(apparent molecular mass of 12 kDa in SDS-polyacrylamide gel
electrophoresis) and in which Cys32 and Cys69
are linked by a disulfide bridge. Unmodified ATAC is a cationic protein
with a pI of 11.35 and is capable of binding to heparin. Some 40% of
ATAC is O-glycosylated within 25 min of synthesis, giving
rise to the appearance of a homogeneous 15-kDa (minor fraction) and a
heterogeneous, terminally sialylated 17-19-kDa (major fraction) protein species in SDS-polyacrylamide gel electrophoresis. The secretion of all ATAC protein variants is completed within 30-40 min
of synthesis. In terms of function, various ATAC protein forms were
consistently ineffective in chemotaxis assays. In contrast, both
purified natural ATAC and a chemically synthesized aglycosyl analog
induced locomotion (chemokinesis) in purified CD4+ and
CD8+ T cell populations at 400 ng/ml.
When screening our collection of 100 human T cell activation genes
(1) for "two-signal" genes with expression restricted to T cells,
we recently identified a novel cDNA, which was designated ATAC (ctivation-induced,
cell-derived, nd hemokine-related) (2).
Induction of ATAC in human T lymphocytes requires
simultaneous stimulation by the phorbol ester
PMA1 (signal 1) and ionomycin (signal 2)
and is suppressed by cyclosporin A (2); it thus resembles the
expression characteristics of a number of lymphokine genes
(e.g. interleukin-2, interleukin-8, and tumor necrosis
factor- In parallel with our studies, the same molecule was independently
identified both in the human (single cysteine motif-1) (9) and murine
(lymphotactin) (6) systems. The predicted structural features of the
murine molecule and its expression pattern and chromosomal location are
analogous to the human counterpart (6). In terms of function,
murine and human lymphotactin was reported to be modestly chemotactic
on several lymphocyte populations (6, 7). In contrast to these
findings, we failed to demonstrate chemotactic effects of ATAC on
a variety of cell types with several forms of the putative secreted
protein (2).
All groups identified ATAC/single cysteine motif-1/lymphotactin at the
cDNA level using methods of reverse genetics, without knowing the
biological role of the molecule. The functional experiments reported to
date were thus based on the hypothesis that mature ATAC is a secreted
molecule and were performed with recombinant proteins assumed to
structurally represent the natural cytokine. Since it has been shown
for several chemokines that their function is strictly dependent on the
correct NH2 terminus (the addition or loss of even a single
amino acid residue can have dramatic functional consequences) (10-12)
and given the difficulty of exactly predicting the putative signal
peptidase cleavage site in the ATAC precursor protein, it was
imperative to determine the exact nature of mature ATAC before
performing further functional studies. This report describes the
purification and structural and biochemical characterization of
secreted ATAC. In addition, we provide initial functional data obtained
with purified natural ATAC and a synthetic analog.
The polymerase chain reaction was used to introduce a
SphI restriction site proximal to Gly23 of human
ATAC cDNA and a HindIII restriction site
distal to the stop codon of the open reading frame. The resulting
SphI-HindIII fragment was subcloned into the
vector pQE (QIAGEN Inc.) to express recombinant fusion proteins in
E. coli M15 according to the manufacturer's instructions.
Denatured recombinant protein was purified on a Ni2+-nitrilotriacetic acid-agarose column (QIAGEN Inc.) and
used for the generation of a goat antiserum.
ATAC was immunoprecipitated from supernatants of nylon
wool-purified lymphocytes or CD8+ T cells activated by PMA
(20 ng/ml; Sigma) and ionomycin (500 ng/ml; Sigma) for 24 h or
from supernatants of transfectant 7.10. For detection of intracellular
ATAC, 5 × 106 cells were lysed in 500 µl of lysis
buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
and 1% Nonidet P-40). Quantitative immunoprecipitations were performed using an excess of ATAC-specific monoclonal antibody
ASA-1 2 or the ATAC-specific antiserum. In
some experiments, brefeldin A (Sigma) was added for the last 4 h
of cell stimulation at 5 µg/ml. Immunoprecipitated proteins were
subjected to SDS-PAGE (13) on 14% gels under reducing conditions
(unless otherwise indicated) using a 6-60-kDa ladder (Life
Technologies, Inc.) as a molecular mass marker, transferred
electrophoretically onto a polyvinylidene difluoride membrane
(Tropifluor, Tropix Inc.), and stained with goat anti-ATAC serum using
the Western Light kit (Tropix Inc.). For carbohydrate analysis,
SDS-PAGE-separated proteins were blotted onto nitrocellulose (BA-S 85, Schleicher & Schuell) and stained using the glycan/protein
double-labeling system (Boehringer Mannheim, 1500783) or the glycan
differentiation system (Boehringer Mannheim, 1210238).
Nylon wool-purified lymphocytes
(5 × 106) were activated with PMA and ionomycin for
4 h, washed, and incubated in cysteine/methionine-deficient RPMI
1640 medium (labeling medium) and 10% dialyzed fetal calf serum for 15 min at 37 °C. Cells were harvested, resuspended in 1 ml of labeling
medium, pulsed with 0.5 mCi of [35S]methionine/cysteine
(1000 Ci/mmol; Tran35S-label, ICN) for 15 min, washed twice
in cold phosphate-buffered saline, and chased for 2-60 min in RPMI
1640 medium and 10% fetal calf serum at 37 °C. Proteins
immunoprecipitated from cell lysates and supernatants with mAb ASA-1
were separated by SDS-PAGE (14% gel), and the gel was fixed and
autoradiographed.
CD8+ T cells
(4-5 × 108) were isolated from nylon wool-treated
peripheral blood mononuclear cells using Magnetobeads to a purity of
>98% and activated at 2 × 106 cells/ml with PMA and
ionomycin for 12-40 h in RPMI 1640 medium supplemented with 5% fetal
calf serum. ATAC was purified from the supernatants using an affinity
column generated by coupling mAb ASA-1 to HiTrap-Sepharose (Pharmacia
Biotech Inc.) according to the manufacturer's instructions. Bound
material was eluted with 100 mM glycine, pH 3.0, and
further purified by microbore Mono S cation-exchange HPLC followed by
reversed-phase RP18 HPLC.
An automated Applied Biosystems Procise sequencer
was used for NH2-terminal sequencing of purified ATAC. The
matrix-assisted laser desorption ionization mass spectrum was recorded
on a REFLEX mass spectrometer (Bruker, Bremen, Germany) equipped with a
N2 laser in linear mode. Sinapinic acid or
ATAC
cDNA containing the entire open reading frame (2) was cloned into
the BCMGSneo vector (14) and transfected into myeloma
P3x63Ag8.653 cells (American Type Culture Collection) by
electroporation. Clone 7.10 was selected for high ATAC
mRNA expression.
ATAC corresponding to the
natural protein was synthesized using solid-phase methods,
HPLC-purified, renatured, and analyzed by mass spectrometry as
described elsewhere (10, 11).
CD4+ and CD8+
cells were positively selected (10 min, 4 °C) using Magnetobeads
coated with mAb 66.1 (CD4, IgM; Dynal, Inc.) or ITI-5C2 (CD8, IgM;
Dynal, Inc.) at a bead/cell ratio of 3:1 and subsequently detached from
the beads (45 min, 20 °C) with polyclonal anti-Fab antibodies
(Dynal, Inc.). Purified cell populations were >99% CD3+
and 96-99% CD4+ or CD8+. Contamination with
CD14+, CD19+, CD37+, and
CD57+ cells and nonspecifically isolated CD4+
or CD8+ cells was <1.5%. Viability of purified T cell
populations was verified by trypan blue exclusion and was always
>97%. Control experiments excluded an influence of the purification
procedure on the migration and phenotype of the analyzed cell
populations (15). T cells (3 × 105) were suspended in
a collagen solution containing 1.6 mg/ml type I bovine dermal collagen
(Vitrogen 100, Collagen Corp., Palo Alto, CA) in Eagle's modified
minimal essential medium, pH 7.4. This suspension was allowed to
polymerize (20-30 min, 37 °C, 5% CO2) in a
self-constructed migration chamber. Immediately after polymerization of
the lattices, natural or synthetic ATAC was added to the migration chamber at varying concentrations. Cell migration was recorded by
time-lapse video microscopy at 37 °C (magnification × 64, detection depth > 200 mm). Stimulation of T cells with PMA, which
induces locomotion in virtually all T cells with little donor
variation, was routinely used as a positive control. Locomotor
base-line activity and stimulation experiments were performed
simultaneously using independent time-lapse units. For evaluation of
cell locomotion, the paths of 30 randomly selected cells were digitized
as x and y coordinates at a 60-s time interval
from step to step using computer-assisted cell tracking as described
previously (15, 16). For evaluation of the dose-response experiments,
the mean percentage of cells locomoting over the entire observation
period in response to phosphate-buffered saline (locomotor base-line activity) was subtracted from the mean percentage of cells locomoting in response to the various concentrations of ATAC dissolved in phosphate-buffered saline.
To determine whether
ATAC is a secreted protein, CD8+ T cells or nylon
wool-purified lymphocytes were stimulated with PMA and ionomycin, and
the cell lysates and supernatants were subjected to immunoprecipitation
with ATAC-specific mAb ASA-1. The immunoprecipitates were separated by
SDS-PAGE and immunoblotted using an antiserum generated against a
recombinant ATAC protein. Whereas no signal could be detected in
lysates and supernatants of unstimulated cells used as controls (Fig.
1A, lanes 1 and 2), a
faint band of ~12 kDa was observed in lysates of activated
lymphocytes (lane 3). With supernatants of activated cells,
a major band of ~12 kDa, a sharp band of 15 kDa, and a diffuse band
ranging from 17 to 19 kDa were obtained (Fig. 1A, lane
4). When brefeldin A, an inhibitor of intracellular transport and
protein secretion (17), was added for the last 4 h of cell
culture, a substantial increase in the 12-kDa signal was detectable in
the cell lysates (Fig. 1A, compare lanes 5 and
3). These results determined that natural ATAC is a protein
secreted by CD8+ T cells upon stimulation. Furthermore, the
experiments demonstrated that mature ATAC is a mixture of several
protein species with differing molecular masses. To exclude any
selectivity of mAb ASA-1 among the forms of natural ATAC, supernatants
of activated CD8+ T cells were also subjected to
immunoprecipitation using an ATAC-specific antiserum (Fig.
1B, lane 2; preimmune serum control, lane
1) in parallel with mAb ASA-1 (lane 3).
To determine the
kinetics of ATAC generation, nylon wool-purified lymphocytes were
activated with PMA and ionomycin for 4 h to achieve maximal
ATAC mRNA levels (2). The cells were pulsed with
[35S]Met/Cys for 15 min and chased for various time
intervals. Within 25 min of synthesis (15-min pulse, 10-min washing),
all ATAC protein species (12, 15, and 17-19 kDa) could be detected in
immunoprecipitates of cell lysates (Fig. 2, lanes
3 and 4). All variants of ATAC could also be detected
in supernatants of activated lymphocytes as soon as 25 min after
synthesis and accumulated thereafter (Fig. 2, lanes 9-13).
Lymphocytes activated with PMA (which alone cannot induce the
ATAC gene (2)) and the corresponding supernatants were used
as specificity controls (Fig. 2, lanes 1 and 2).
The analysis of all cell lysates (Fig. 2, lanes 3-8) and
supernatants (lanes 9-13) of PMA- and ionomycin-activated
lymphocytes revealed that ATAC is secreted within ~30-40 min of
synthesis.
CD8+ lymphocytes
were stimulated for up to 24 h with PMA and ionomycin, and ATAC
was affinity-purified from the culture supernatants with mAb ASA-1
coupled to Sepharose. When the affinity-bound material was subjected to
cation-exchange HPLC, the bulk of the 12- and 15-kDa ATAC species was
identified in fraction 12 by immunoblotting (Fig.
3A) and further purified to homogeneity (as
determined by SDS-PAGE; data not shown) by reversed-phase HPLC (Fig.
3B and Fig. 4A). Natural ATAC
could also be purified by reversed-phase HPLC following enrichment on a
heparin column (data not shown).
Although our initial purification of ATAC was
accompanied by a significant loss of the 17-19-kDa species and a
relative reduction of the 15-kDa species (Fig. 4A,
lane 1), the material was well suited for the
characterization of the protein backbone of ATAC since the comparison
with a chemically synthesized ATAC protein (Val22-Gly114) (Fig. 4A, lane
2) indicated that the 12-kDa species is a post-translationally unmodified form of ATAC. This comparison also suggested that the 15- and 17-19-kDa variants (see also Figs. 1 and 2) represent post-translational modifications of the 12-kDa ATAC protein
species.
Automated Edman degradation of the purified ATAC preparation shown in
Fig. 4A determined Val22 as the
NH2-terminal amino acid residue. This result indicated that
mature ATAC is generated from the precursor molecule by proteolytic cleavage between Gly21 and Val22. When the
purified ATAC preparation was subjected to matrix-assisted laser
desorption ionization mass spectrometry (Fig. 4B) and
electrospray mass spectrometry (data not shown), the mass obtained
(10,272.1 Da) corresponded well to the calculated 10,271.72 Da for an
unmodified Val22-Gly114 ATAC protein. Thus, an
estimated 60% of natural ATAC ("12-kDa species," 10,271.72 Da;
compare Figs. 1 and 2) is secreted as an unmodified protein of 93 amino
acids (Fig. 4C). For reasons given in the legend to Fig. 4,
the 15-kDa species present in the preparation did not give a signal in
mass spectrometry.
In matrix-assisted laser desorption ionization analysis (Fig.
4B) and electrospray mass spectrometry (data not shown), a
smaller second peak with a mass of 10,116.9 Da was observed. Since
Edman degradation of two independently purified ATAC preparations did not reveal contaminating proteins or the presence of
NH2-terminally truncated ATAC protein variants, this peak
possibly resulted from the presence of an ATAC variant lacking the
COOH-terminal amino acid residues Thr113 and
Gly114 (calculated molecular mass of 10,113.56 Da).
To further characterize the nature of the observed 15- and
17-19-kDa proteins, we transfected myeloma P3x63Ag8.653 cells with a cDNA containing the entire coding region of ATAC (2).
When supernatants of transfectant 7.10 were analyzed by
immunoprecipitation with mAb ASA-1 followed by immunoblotting, the 12-, 15-, and 17-19-kDa protein species identified corresponded to the
proteins immunoprecipitated from supernatants of activated lymphocytes
(Fig. 5A, compare lanes 1 and
2). No signal was obtained with supernatants of the
nontransfected control myeloma cells (data not shown). The 15- and
17-19-kDa protein species could thus be determined as products of the
ATAC gene, which arise by post-translational modification of
the Val22-Gly114 ATAC protein (12-kDa
species). Further analysis demonstrated that both the 15- and
17-19-kDa proteins are glycosylated (Fig. 5B). In the
17-19-kDa protein, the presence of terminal sialic acid
Mature ATAC contains only two cysteines, at
positions 32 and 69. To test for disulfide bonding between these
cysteines, ATAC was immunoprecipitated from supernatants of activated
lymphocytes, separated in parallel by SDS-PAGE under reducing (Fig.
6, lanes 1 and 3) and nonreducing
(lane 2) conditions, and analyzed by immunoblotting. The
altered mobility of the unfolded protein in the reduced state as
compared with its mobility in the nonreduced state indicated the
presence of a disulfide bond in the mature ATAC protein.
CD4+ and CD8+
peripheral blood T cells were purified and tested in a collagen matrix
chemokinesis assay for increased locomotion using time-lapse video
microscopy. In all experiments, chemically synthesized ATAC
(Val22-Gly114) was used at 400 ng/ml
(determined to be the optimal concentration after preliminary
titrations). In several experiments, preparations of purified natural
ATAC containing both the nonglycosylated and glycosylated forms of the
cytokine in the correct relative amounts (compare Fig. 1) were used in
parallel. With CD4+ T cells from different blood donors, a
substantial increase in the percentage of migrating cells could be
observed in approximately one-third of the experiments (positive
results are shown in Fig. 7, A-D). In
another one-third of the experiments, there was a modest increase in
locomotion; the remaining experiments were negative. The results
obtained with purified CD8+ T cells were similar. Again, in
approximately one-third of the experiments, a clear increase in
locomotion was recorded (positive results are shown in Fig. 7,
E-H); in another one-third of the experiments, a modest
response was observed. When tested in parallel, the effect of natural
ATAC was more pronounced when compared with the synthetic ATAC protein
(Fig. 7, A and E). The specificity of the
chemokinetic effect of natural ATAC on CD4+ T cells (Fig.
7C) and CD8+ T cells (Fig. 7G) was
verified with an antiserum raised against the ATAC protein.
Representative dose-response effects of ATAC on CD4+ and
CD8+ T cells are shown in Fig. 7 (D and
H). In a number of donors, only one T cell subset responded
to ATAC by increased chemokinesis.
In terms of structure, we have demonstrated in this work that
natural ATAC is a secreted protein of 93 amino acids that is generated
from the precursor molecule (2, 7, 9) by proteolytic cleavage between
Gly21 and Val22. Some 60% of ATAC is secreted
as an unmodified cationic protein (pI 11.35) with a molecular mass of
10,271.7 Da, running in SDS-PAGE with an apparent molecular mass of 12 kDa. A substantial proportion of ATAC is rapidly glycosylated and
secreted within 30-40 min of synthesis; these glycosylated forms of
ATAC can be identified by SDS-PAGE as 15-kDa (minor fraction) and
17-19-kDa (major fraction) proteins. Finally, we could determine that
the two cysteines present in the ATAC protein are linked by a disulfide
bridge.
Considering the structural aspects, ATAC thus exhibits both
similarities to and differences from chemokines of the CC and CXC families, to which it is related by its protein sequence
(2, 7-9). It shares with these chemokines a disulfide bridge between the two cysteines conserved in all of these molecules. However, ATAC
lacks the other pair of cysteines and the second disulfide bond and
thus must have a clearly different tertiary structure compared with the
CC and CXC molecules (2, 18). Another conspicuous finding is
the comparatively long COOH terminus of ATAC. This finding is relevant,
given the observation that modifications of the COOH terminus can be
critical for the function of chemokines (10). Unlike CXC
chemokines, which are not glycosylated (19), a substantial proportion
of ATAC is O-glycosylated and thus resembles MCP-1 (19-21).
The functional relevance of the observed glycosylation is unclear at
present since the aglycosyl form of ATAC is functional in chemokinesis
assays with T cells (see below). The cationic nature of the secreted
aglycosyl form of ATAC and its observed binding to heparin in
vitro (data not shown), features shared with the CC and
CXC chemokines, potentially enable ATAC to bind to
glycosaminoglycans of the tissue matrix and cell membranes and thus to
persist in the microenvironment for a prolonged period of time (22,
23).
In terms of function, we could demonstrate that natural ATAC, as well
as its nonglycosylated form, can induce substantial chemokinesis in
primary CD4+ and CD8+ human T cells. When
effective, ATAC induced a similar proportion of T cells to locomote, as
did interleukin-8 in our previous experiments (16). For as yet unknown
reasons, the effect of ATAC on T cells was clearly
donor-dependent (a systematic analysis of this donor dependence will require the identification of the ATAC receptor). More
important, when chemically synthesized ATAC was tested in parallel with
purified natural ATAC, the results were always concordant. Our findings
thus differ from the negative results of the chemokinesis assays
reported by Kelner et al. (6), who tested murine
lymphotactin for effects on CD4+-depleted,
CD8+-depleted, and double-negative murine thymocytes using
a filter-based assay system. The interpretation of these divergent
results is difficult since both the populations tested and the assay
systems used differed considerably; in addition, the assays with murine cells were performed with a form of lymphotactin that probably represents a +1 NH2-terminal variant.
Our numerous attempts to induce chemotaxis with ATAC in various cell
systems have remained unsuccessful. We have previously reported
negative results obtained with two ATAC molecules,
Gly21-Gly114 (+1 NH2-terminal
variant) and Gly23-Gly114 ( We greatly appreciate the expert technical
assistance of Birgit Radke and Katja Ranke.
Molecular Immunology,
Institute of Biochemistry,
Biomedical Research Center and
Department of Biochemistry and Molecular Biology, Vancouver, British
Columbia, V6T 1Z3 Canada
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) (3-5). The open reading frame of ATAC encodes a
protein of 114 amino acids with an overall structural similarity to
chemokines of the CXC and CC families (2). However, several
findings suggest that ATAC represents a new class of cytokine: there is
only one cysteine at the corresponding protein locus, indicating a
tertiary structure different from the known chemokines, and the
selective expression of ATAC in CD8+ T cells and
CD8+ thymocytes (2, 6, 7) contrasts with the
characteristically broad expression pattern of classical chemokines
(8). In addition, the location of the ATAC gene on human
chromosome 1q23 (2, 7) differs from CXC and CC chemokine
genes, which are clustered on chromosomes 4 and 17, respectively
(8).
Expression of ATAC (Gly23-Gly114) in
Escherichia coli and Generation of an ATAC-specific Goat
Antiserum
-cyano-4-hydroxycinnamic acid was used as the matrix (saturated
solution in aqueous 0.1% trifluoroacetic acid/acetonitrile at a ratio
of 2:1). The protein was dissolved in aqueous 0.1% trifluoroacetic
acid/acetonitrile (2:1) and mixed 1:1 with the matrix solution. The
electrospray ionization mass spectrum was obtained on a TSQ 700 quadrupole mass spectrometer (Finnigan MAT, Bremen, Germany) equipped
with an electrospray ion source. ATAC was dissolved in
methanol/water/acetic acid (50:50:1) and introduced into the ion source
by a microsyringe pump (Harvard Apparatus Ltd.) at a flow rate of 0.5 µl/min.
Natural ATAC Is Secreted by Activated CD8+ T Cells as
12-, 15-, and 17-19-kDa Protein Species
Fig. 1.
Natural ATAC is secreted by activated
CD8+ lymphocytes as 12-, 15-, and 17-19-kDa protein
species. A, forms of natural ATAC. Using mAb ASA-1, ATAC was
immunoprecipitated from lysates (Lys; lanes 1,
3, and 5) and supernatants (Sup;
lanes 2 and 4) of nylon wool-purified
CD8+ lymphocytes, which either were left unstimulated
(lanes 1 and 2) or were stimulated with PMA
(P) and ionomycin (I) for 24 h (lanes
3-5). In some experiments, brefeldin A (Bref.
A; 5 µg/ml) was added for the last 4 h of cell culture
(lane 5). Immunoprecipitated proteins were separated by
SDS-PAGE (14% gel), blotted onto a polyvinylidene difluoride membrane,
and probed with an ATAC-specific antiserum. B, comparison of
ATAC forms immunoprecipitated by an ATAC-specific antiserum and mAb
ASA-1. ATAC was immunoprecipitated from supernatants of activated
CD8+ T cells with an immune serum (IS;
lane 2) and in parallel with mAb ASA-1 (lane 3).
A preimmune serum control was also included (pre-IS;
lane 1). Immunoblotting was performed as described for A.
[View Larger Version of this Image (35K GIF file)]
Fig. 2.
Kinetics of ATAC generation and
secretion. Nylon wool-purified lymphocytes were stimulated with
PMA (P) and ionomycin (I) for 4 h,
pulse-labeled for 15 min using [35S]cysteine/methionine,
and chased for 0-60 min. Generation and secretion of various ATAC
protein species were tracked by quantitative immunoprecipitation from
cell lysates (Lys; lanes 3-8) and culture supernatants (Sup; lanes 9-13) using mAb ASA-1.
Immunoprecipitated proteins were separated by SDS-PAGE (14% gel) and
visualized by autoradiography. Immunoprecipitates from lymphocytes
stimulated with PMA only (no generation of ATAC) were used as
specificity controls (lane 1, cell lysate; lane
2, culture supernatant).
[View Larger Version of this Image (57K GIF file)]
Fig. 3.
Purification of natural ATAC. A,
cation-exchange HPLC of ATAC. ATAC was purified from supernatants of
PMA- and ionomycin-activated CD8+ lymphocytes using an
affinity column (mAb ASA-1 coupled to HiTrap-Sepharose). Bound material
was eluted with 100 mM glycine, pH 3.0, and further purified by microbore Mono S cation-exchange HPLC. Proteins were eluted
with a gradient of increasing concentrations of NaCl in ammonium
formate, pH 4.0, containing 25% (v/v) acetonitrile. ATAC was
identified in fractions (Fr.) 11-13 by immunoblotting
(inset). B, reversed-phase HPLC of ATAC. For the
characterization of the protein backbone of ATAC, the 12- and 15-kDa
species were purified to homogeneity by reversed-phase RP18 HPLC of
fraction 12. The protein that eluted as a single peak at 43%
acetonitrile (single broad band in SDS-PAGE; data not shown) was
verified as the 12- and 15-kDa ATAC species by immunoblotting (see Fig.
4A).
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
NH2-terminal protein sequencing
and mass spectrometry of natural secreted ATAC. A,
comparison of a purified ATAC preparation with a chemically synthesized
ATAC protein (Val22-Gly114). Purified ATAC was
analyzed by SDS-PAGE (14% gel) and immunoblotting (purif.;
lane 1) in parallel with a chemically synthesized ATAC protein (Val22-Gly114) (synth.;
lane 2). B, matrix-assisted laser desorption
ionization/time-of-flight spectrum of the same ATAC preparation (with
-cyano-4-hydroxycinnamic acid as the matrix). The spectrum shows the
[M + H]+ (m/z 10273.1) and [M + 2H]2+ (m/z 5137.1) peaks of ATAC and the [M + H]+ (m/z 10117.9) and [M + 2H]2+
(m/z 5060.0) peaks of a minor component. The 15-kDa form of
purified ATAC observed in the immunoblot (A) and later
identified as a glycosylated variant was not detectable in the mass
spectrometry experiment since the matrices used
(
-cyano-4-hydroxycinnamic acid and sinapinic acid) are not suitable
for the analysis of glycoproteins. a.i., arbitrary
intensity. C, schematic representation of secreted mature
ATAC in comparison with the precursor molecule. Indicated is the
proteolytic cleavage site between Gly21 and
Val22 as determined by NH2-terminal sequencing
of the mature protein. The hydrophobic signal sequence of the precursor
molecule is shown (black box). Numbering of amino acids
(aa) is according to the protein sequence of the precursor
molecule as deduced from ATAC cDNA (2).
[View Larger Version of this Image (15K GIF file)]
(2-3)-bound to galactose could be detected (Fig. 5C) (a variable degree of glycosylation is apparently responsible for the
observed diffuse nature of this ATAC species). Since the ATAC protein
sequence does not contain any consensus sites for N-linked glycosylation, the 15- and 17-19-kDa species could thus be identified as O-linked glycosylation forms of mature ATAC.
Fig. 5.
The 15- and 17-19-kDa species are
O-glycosylated variants of the ATAC protein. A,
shown is a comparison of proteins immunoprecipitated from supernatants
of an ATAC transfectant and from supernatants of activated
CD8+ T cells using ATAC-specific mAb ASA-1. Supernatants of
ATAC transfectant 7.10 (lane 1) and supernatants
of PMA- and ionomycin-activated CD8+ T cells (lane
2) were immunoprecipitated and immunoblotted in parallel using mAb
ASA-1 and an ATAC-specific antiserum. B, the 15- and
17-19-kDa ATAC species are glycosylated. Supernatants of activated
lymphocytes were subjected to immunoprecipitation with mAb ASA-1 and
blotted onto nitrocellulose. The blotted proteins were identified as
glycoproteins by specific labeling of carbohydrate hydroxyl groups
using periodate oxidation. C, the 17-19-kDa ATAC species
contains terminal sialic acid (2-3)-bound to galactose. The binding
of the lectin Maackia amurensis agglutinin indicated the
presence of terminal sialic acids
(2-3)-bound to galactose in the
17-19-kDa ATAC species. No binding of the lectins Galanthus nivalis agglutinin, Sambucus nigra agglutinin, peanut
agglutinin, and Datura stramonium agglutinin was observed
with the 15- or 17-19-kDa species (data not shown). NTC,
nylon wool-purified T cells.
[View Larger Version of this Image (35K GIF file)]
Fig. 6.
Mature ATAC contains a disulfide bridge.
ATAC was immunoprecipitated from supernatants of activated lymphocytes
using mAb ASA-1. Precipitated proteins were eluted under reducing
(R; lanes 1 and 3) or nonreducing
(NR; lane 2) conditions, separated by SDS-PAGE
(14% gel), and immunoblotted using an ATAC-specific antiserum. The
reduced form of ATAC was electrophoresed in two separate lanes
(lanes 1 and 3) to provide an optimal comparison with the nonreduced form.
[View Larger Version of this Image (61K GIF file)]
Fig. 7.
Locomotion of CD4+ and
CD8+ T cells in response to natural and synthetic ATAC
proteins. Purified CD4+ and CD8+ T cells
were suspended in a collagen matrix, and natural (nat.) or
synthetic (syn.) ATAC
(Val22-Gly114) was added at 400 ng/ml. Control
experiments were set up in parallel. Time-dependent changes
in T cell locomotion were recorded using time-lapse video microscopy.
Experiments with CD4+ and CD8+ T cells are
shown in A-D and E-H, respectively. A direct
comparison of the effects of natural and synthetic ATAC proteins is
shown in A and E. The chemokinetic effect of
purified natural ATAC on CD4+ T cells (C) and
CD8+ T cells (G) was inhibited by an
ATAC-specific antiserum (1:100 dilution), but not by a preimmune serum
at the same dilution (data not shown). Dose-response effects of
synthetic ATAC (Val22-Gly114) were tested on
CD4+ and CD8+ T cells of three responders;
representative results are shown in D and H.
Locomotor base-line activity in response to an equivalent volume of
phosphate-buffered saline (solvent for ATAC) is indicated in all
experiments (control).
[View Larger Version of this Image (31K GIF file)]
1
NH2-terminal variant), both generated as fusion proteins and also by chemical synthesis and which were tested on unseparated T
cells, purified CD4+ and CD8+ T cells, as well
as CD4+ and CD8+ T cell clones (2). Although
neither of the two ATAC variants truly represents the natural molecule,
as this work has shown, we know from the chemokinesis assays that the
1 NH2-terminal ATAC variant is
functional.3 Our chemotaxis assays with
CD4+ and CD8+ T cells and T cell clones,
monocytes, and neutrophils performed earlier with supernatants of the
ATAC transfectant 7.10 (compare Fig. 5A) (ATAC at
1 µg/ml to 1 pg/ml) were negative as well (2). New chemotaxis
experiments performed with purified natural ATAC (containing all forms
of the molecule) on primary human T cells and also T cells
prestimulated with phytohemagglutinin and interleukin-2 for 7 days
again gave no conclusive results, although the cells were responsive to
MCP-1.4 Thus, our data regarding the
chemotactic capabilities of ATAC differ from the studies of Kelner
at al. (6) and Kennedy et al. (7), which were
performed on murine and human cells using murine (presumed +1
NH2-terminal variant) and human lymphotactin, respectively.
The reasons for this discrepancy are unclear at present since the test
systems used by these investigators were similar to ours. Further
experiments will be necessary to determine whether ATAC can
functionally be grouped into the family of chemokines.
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant Kr827/11-1 (to R. A. K.) and Grant Ch38/7-2 (to J. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶¶
To whom correspondence should be addressed: Molecular
Immunology, Robert Koch Inst., Nordufer 20, 13353 Berlin, Federal
Republic of Germany. Tel.: 49-30-4547-2450; Fax: 49-30-4547-2603.
1
The abbreviations used are: PMA, phorbol
12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; mAb,
monoclonal antibody; HPLC, high-performance liquid
chromatography.
2
S. Müller, R. A. Kroczek, manuscript in
preparation.
3
F. Entschladen and R. A. Kroczek, unpublished
observations.
4
B. Dorner and R. A. Kroczek, unpublished
observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.