Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.
SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 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-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus 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.
Oliva
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AA
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637, USA.
Steiner
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DF
Chan
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D005075:Biological Evolution; D002816:Chordata, Nonvertebrate / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D007328:Insulin / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013002:Somatomedins / Q000032:analysis; D013045:Species Specificity; D013381:Subtilisins / Q000235:genetics*; D014018:Tissue Distribution
D018076:DNA, Complementary; D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; D013002:Somatomedins; D043484:Proprotein Convertases; D013381:Subtilisins; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases