Isabella Cascino, Giuliana Papoff, Adriana Eramo, and Giovina Ruberti

Department of Immunobiology, Institute of Cell Biology, National Research Council, Rome, Italy.

Received 12/01/95; Accepted 01/11/96; On-line 1/1/96

Several of the variants depicted in Fig. 1 retain the hydrophobic leader peptide but lack the hydrophobic transmembrane domain. This suggests that they might be expressed as soluble forms and secreted. Moreover they share the 5' portion (exons 1 and 2) corresponding to the N-terminal 49 amino acids of the mature protein. This may indicate that this is a physiologically important domain and that, in turn, these splicing variants must have some defined functional activity. However, before considering this possibility, it was important to demonstrate that these mRNA variants could be translated as proteins and, more importantly, that they could be secreted in the extra-cellular fluid. This initially presented some problems because several Fas monoclonal antibodies i.e. CH-11 (Upstate Biotechnology Inc., Lake Placid, NY) and DX2 (42) reacted with Fas and FasExo6Del but did not react with the other variants (29). The most likely explanation is that these antibodies must recognise a sequence contained in exons 4 and/or 5, since these are the only two extracytoplasmic regions that are missing in all the Ab-negative variants. After a rather extensive search three antibodies, M24, M1 (43) and FasN18 (Santa Cruz, Biotechnology,CA) which are capable of recognizing the 49 amino acids at the N-terminal region were identified (30). A sandwich ELISA using M24 and FasN18 antibodies was used to detect the Fas variants in both cell lysates and supernatants of transfected cells. Fas proteins were found to be present in the supernatants at a concentration two orders of magnitude higher than that found intracellularly, suggesting that the Fas isoforms code for Fas soluble proteins and that these are secreted by the cells and accumulate in the medium (30).