Two Forms of the Apoptosis-linked Protein ALG-2 with Different Ca super(2+) Affinities and Target Recognition

The apoptosis-linked gene ALG-2 encodes a Ca super(2+)-binding protein of the penta EF-hand family. To investigate the Ca super(2+) binding properties of the recombinant ALG-2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521- 525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly super(121) and Phe super(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca super(2+). In the presence of Tween the Ca super(2+) binding profiles display two high affinity sites with [Ca super(2+)] sub(0.5) values of 1.2 and 3.1 mu M for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2,5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca super(2+) binding properties and function of the proteins.