Elephantfish proinsulin possesses a monobasic processing site.
Total pancreatic RNA from the holocephalan species Callorhyncus milii (elephantfish) was used to make cDNA as a template for the polymerase chain reaction. Three redundant primers based on the known amino acid sequence of elephantfish insulin were used to amplify a fragment of proinsulin comprising truncated B-chain, complete C-peptide, and complete A-chain. Whereas the C-peptide/A-chain junction contained the expected dibasic cleavage site (-Lys-Arg-), the B-chain/C-peptide junction was found to contain only a single Arg, the first such site to be unequivocally associated with the proteolytic processing of a proinsulin to insulin. Examination of the flanking sequences around this site shows that a typical endocrine/neuroendocrine PC3 conversion enzyme should still be able to cleave, as the general requirements for precursor processing at a monobasic site are satisfied, notably a basic residue (Lys) at the -4 position. An acidic residue (in this case Asp) at the +1 position, which is seen in all known proinsulins, is maintained. The corresponding genomic DNA fragment of elephantfish proinsulin was also amplified by PCR, revealing a 402-bp intron at the conserved IVS-2 position within the C7 codon.
Gieseg
M A
MA
School of Medical Sciences, University of Otago, Dunedin, New Zealand.
Swarbrick
P A
PA
Perko
L
L
Powell
R J
RJ
Cutfield
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002096:C-Peptide / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D005399:Fishes; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics; D012313:RNA; D017385:Sequence Homology; D013698:Templates, Genetic
D002096:C-Peptide; D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D011384:Proinsulin