Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein-Barr virus vector.
Precursors of most secreted and cell surface molecules carry signal sequences at their amino termini. Here we describe an efficient signal sequence trap method and isolation of a novel CC chemokine. An expression library was constructed by inserting 5' portion-enriched cDNAs from phytohemagglutinin-stimulated peripheral blood mononuclear cells into upstream of signal sequence-deleted CD4 cDNA in an Epstein-Barr virus shuttle vector. After electroporation into Raji cells, CD4 antigen-positive cells were enriched by repeated cell sorting and plasmids were recovered in Escherichia coli. Out of 100 plasmid clones examined, 42 clones directed expression of CD4 antigen on the cell surface. Among them were signal sequences of CD6, beta2-microglobulin, MGC-24, and T cell receptor epsilon-chain, and at least four novel potential signal sequences. A cDNA clone encoding a novel CC chemokine was isolated by using one of the trapped fragments. The gene designated as TARC from Thymus and Activation-Regulated Chemokine was expressed transiently in phytohemagglutinin-stimulated peripheral blood mononuclear cells and constitutively in thymus. Radiolabeled recombinant TARC specifically bound to T cell lines and peripheral T cells but not to monocytes or granulocytes. The binding of radiolabeled TARC to the high-affinity receptor (Kd, 2.1 nM) on Jurkat was displaced by TARC but not by interleukin-8, MIP-1alpha, RANTES, or MCP-1. TARC also bound to the promiscuous chemokine receptor on erythrocytes (Kd, 17 nM). TARC induced chemotaxis in T cell lines Hut78 and Hut102. Pretreatment of Hut78 with pertussis toxin abolished the TARC-induced cell migration. Collectively, T cells express a highly selective receptor for TARC that is coupled to pertussis toxin-sensitive G-protein. TARC may a factor playing important roles in T cell development in thymus as well as in trafficking and activation of mature T cells.
Imai
T
T
Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan.
Yoshida
T
T
Baba
M
M
Nishimura
M
M
Kakizaki
M
M
Yoshie
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D018925:Chemokines / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism*
D018925:Chemokines; D017931:DNA Primers; D018076:DNA, Complementary; D010835:Phytohemagglutinins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins