Multiple rat brain calpastatin forms are produced by distinct starting points and alternative splicing of the N-terminal exons.
5'-RACE was performed on rat brain calpastatin mRNA and two new translation initiation ATG's were found. The first one is upstream of the previously designed initiation translation site localized in the rat calpastatin L-domain. The deduced protein sequence of this region is highly homologous to the XL-domain of calpastatin type I in other species. The other ATG has not previously been reported and is localized in exon 8, thus originating a calpastatin isoform constituted only by four repetitive inhibitory units without the XL-L-domains. Transcripts from the rat brain calpastatin gene are also subjected to multiple splicing events involving exons 4, 6, 8 in different combinations. A series of recombinant calpastatin forms was produced that differed in the exons present in the L-domain, and all the variants showed comparable inhibitory efficiency against calpain. It was concluded that the presence of the XL-domain in these isoforms is not relevant for the formation of the calpain/calpastatin complex in the absence of calcium, that is the interaction of calpastatin with inactive calpain. Using exon-specific antisera, specific calpastatin protein isoforms containing the XL-domain have been detected in rat brain homogenates.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T2 T3 T4 T5 T6 T7 T8 T9
GENE-N GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-N GENE-N GENE-N GENE-Y CHEMICAL GENE-Y CHEMICAL GENE-Y GENE-N GENE-Y GENE-N GENE-N GENE-Y GENE-N
calpastatin type I calpastatin XL-L-domains rat brain calpastatin calpastatin L-domain calpain XL-domain calpain calpastatin calcium rat brain calpastatin N calpastatin calpain calpastatin XL-domain rat calpastatin L-domain rat brain calpastatin XL-domain