We have cloned a 2.4-kilobase cDNA from a human placental cDNA library by using a y-glutamyl transpeptidase [GGT; y-glutamyltransferase, (5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] probe. The deduced amino acid sequence of this cDNA, GGT-rel, exhibited an overall similarity of 39.5% with human GGT. Sequences that could represent a heavy and a light chain, analogous to GGT, as well as a putative transmembrane region were identified in GGT-rel. Transfectants overexpressing GGT-rel were tested for their ability to catalyze cleavage of the y-glutamyl moiety from natural and synthetic substrates for GGT. Experiments with glutathione added to the medium suggested that GGT-rel could hydrolyze the r-glutamyl moiety.More definitive evidence was obtained in experiments in which this protein converted leukotriene C4 to leukotriene D4. However, GGT-rel did not convert synthetic substrates that are commonly used to assay GGT. Our results indicate that GGT can no longer be considered the only enzyme capable of cleaving the y-glutamyl linkage of leukotriene C4 and, most likely, of other natural compounds.y-Glutamyl transpeptidase [GGT; y-glutamyltransferase, (5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] is one of the major enzymes involved in the metabolism of glutathione (L-y-glutamyl-

X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma- cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral- mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.

A variant of severe combined lmmunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4 + cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4 + T cell lines w.ere established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SClD may be corrected by gene therapy.

Engagement of the high-affinity IgG Fc receptor (FcγRI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in FcγRI-mediated signaling. Cross-linking of the FcγRI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the FcεRIγ subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with FcεRIγ after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.