PROPROTEIN CONVERTASES IN AMPHIOXUS - PREDICTED STRUCTURE AND EXPRESSION OF PROTEASES SPCB AND SPC3

SPC2 and SPC3 are two members of a family of subtilisin-related protea ses which play essential roles in the processing of prohormones into t heir mature forms in the pancreatic B cell and many other neuroendocri ne cells. To investigate the phylogenetic origins and evolutionary fun ction of SPC2 and SPC3 we have identified and cloned cDNAs encoding th ese enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate, The amino acid sequence of preproSPC2 contains 689 aa and i s 71% identical to human SPC2. In contrast, amphioxus preproSPC3 consi sts of 774 aa and exhibits 55% identity to human SPC3, These results s uggest that the primary structure of SPC2 has been more highly conserv ed during evolution than that of SPC3. To further investigate the func tion(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked pol ymerase chain reaction (RT-PCR) assay, Whole amphioxus was dissected l ongitudinally into four equal-length segments and RNA was extracted. U sing RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of S PC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2- to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SP C3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a h omologue of mammalian insulin/insulin-like growth factor, was expresse d predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various am phioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.