The conformational alteration of the mutated extracellular domain of Fas in an adult T cell leukemia cell line

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Abstract

Fas (APO-1/CD95) is a cell surface receptor involved in apoptosis. Almost all adult T cell leukemia (ATL) cells express abundant Fas antigen and show apoptosis induced by IgM anti-Fas monoclonal antibody (mAb). We established the ATL cell line, RSO4, which was obtained from Fas-sensitive ATL cell line SO4 and showed resistance to apoptosis induced by the mAb. By sequencing analysis of Fas gene, we found the mutation with the transition of A–G at nucleotide 373 at exon 2 among the extracellular domain (ECD), resulting in substitution of arginine for histidine. The molecular modeling suggested the definitive conformational alteration around residues 52–58 among the cysteine-rich domain (CRD) 1. It was suggested that the polymerization of Fas antigen, which was the essential process for the efficient induction of apoptosis, was interfered by the alteration of CRD1, and that this portion, named the “histidine-rich region,” played a critical role in Fas assembly.

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Materials and methods

Cell lines. The ATL cell line, SO4, used for the present study was established from the patient's peripheral blood ATL cells, and was of primary ATL cell origin, which was confirmed by the analysis of the integration pattern of HTLV-1 proviral DNA and the rearrangement profile of T cell receptor (TCR) β-chain gene. Another ATL cell line RSO4 was the subline obtained from SO4 by the culture with IgM anti-Fas mAb. These two cell lines were maintained in RPMI 1640 medium containing 10% FCS with

RT-PCR analysis of Fas mRNA

The expected two main products, 1167 and 1104 bp bands, which corresponded to membrane Fas and soluble Fas, respectively, were similarly observed in two ATL cell lines and Jurkat cells, which were used as positive control (Fig. 1). In addition to the two main products, some faint bands were visible at 1000 bp or less in all samples, which corresponded to soluble Fas variants reported by Papoff et al. [37].

Sequencing analysis of RT-PCR products of Fas mRNA

The sequence analysis of full-length RT-PCR products from SO4 cells showed only the

Discussion

Interestingly, human Fas antigen contains “histidine-rich region” at residues 53–60 in cysteine-rich domain (CRD) 1, which are unusual and seen only in the primate Fas antigen (Fig. 3). The two amino acid residues, Tyr and Cys, corresponding to the two histidine sites (His53 and His60) in human Fas antigen, are highly conserved not only among TNF receptors but also among Fas antigens. In human TNF receptor, Tyr49 and Cys59 form the hydrophobic core of CRD1 and a disulfide bond with Cys72,

Acknowledgements

We are grateful to Miss T. Hayashi and Miss N. Dateki for their excellent technical assistance. IL-2 was generously donated by Takeda Pharmaceutical.

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      This mutation is at the position directly upstream to an imperfect N/D-T-V/D-C motif, M-D-A-V-C at amino acids 196–200 corresponding to CRD4 of GenBank TNFRSF1B. There is also a point mutation resulting in substitution of an arginine for a histidine at amino acid 60 of CRD1 of Fas, 20 amino acids upstream of the terminal cysteine, which has been predicted to have a similar effect on stability of CRD1 to defects in the conserved terminal motif which is at amino acids 80–82 in this protein [25]. There are many other mutations affecting CRDs, such as deletions of exons containing CRDs in Fas, described by Fisher et al. [14], which are associated with lymphoproliferative syndromes, but these are not specific point mutations in or near the N/D-T-V/D-C sequences flanking cysteines of CRDs, but rather are global defects in large regions including CRDs.

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