Purification, structural analysis, and function of natural ATAC, a cytokine secreted by CD8(+) T cells.
Recently, we identified a novel putative human cytokine expressed by activated CD8(+) T cells, which was designated ATAC (activation-induced, T cell-derived, and chemokine-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.
Dorner
B
B
Molecular Immunology, Robert Koch-Institute, 13353 Berlin, Federal Republic of Germany.
Müller
S
S
Entschladen
F
F
Schröder
J M
JM
Franke
P
P
Kraft
R
R
Friedl
P
P
Clark-Lewis
I
I
Kroczek
R A
RA
D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002634:Chemotaxis, Leukocyte; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000737:chemistry / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D010449:Peptide Mapping
D002352:Carrier Proteins; D016207:Cytokines; D006023:Glycoproteins