PC8 [corrected], a new member of the convertase family

Abstract

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23-11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Gene organization and alternative splicing of human prohormone convertase PC8

“…Sense strand oligonucleotide oPC72 (5h-GCCTTTGCTGAG-TCAACACTAC-3h), corresponding to nt j54 to j75 of human PC8 mRNA, and anti-sense strand oligonucleotide oPC71 (5h-AGGGAACCAGTAAGAAGAGCC-3h), corresponding to nt j258 to j278 of human PC8 mRNA [19], were used to screen a commercial human P1 genomic library for the hPC8 gene. Three independent P1 clones were obtained from Genome Systems.…”

“…Poly(A + ) RNA (1 µg) from the BEN cell line was reversetranscribed with SuperScript II reverse transcriptase by using PC8-specific anti-sense strand oligonucleotide oPC34 (5h-TACC-CGGATACCTGCGAT-3h), complementary to nt j941 to j958 of human PC8 [19]. Resultant cDNA transcripts were Ctailed with terminal dideoxytransferase (TdT) and PCR amplification was performed with the supplied sense strand 5h RACE adapter primer (Life Technologies) and anti-sense strand PC8-specific oligonucleotide oPC79 (5h-CTGGATTCTACATGA-GGTTGAG-3h), corresponding to nt j145 to j166 of human PC8 mRNA (Figure 3).…”

“…The mammalian members of the prohormone convertase family include furin, also known as PACE [2][3][4][5][6] ; PC2 [7,8] ; PC1, also identified as PC3 [8][9][10][11] ; PC4 [12,13] ; PC5, also known as PC6 [14][15][16] ; PACE4 [17], also identified as PC7 [18] ; and most recently PC8, also referred to as LPC, rPC7 and SPC7 [19][20][21][22]. Furin, PACE4, PC5\6 and PC8 are expressed in a wide variety of cell lines and tissues, although variation in the relative abundance of these enzymes is observed [6,14,15,17,19,21,23]. In comparison, PC1 and PC2 have a more restricted distribution, primarily in cells of endocrine and neuroendocrine origin [7,[8][9][10][11], and PC4 is restricted to the testis [12,13].…”

“…Northern hybridization analysis performed on poly(A) + RNA from a variety of rat tissues and human cell lines demonstrated at least two transcripts for PC8 : a 3.5 kb transcript and a 4.3 kb transcript [19] ; evidence for a larger transcript of approx. 6 kb in several rat tissues has been reported [21].…”

“…The nucleotide sequence of the 5h-flanking region of the human PC8 gene was determined directly by double-stranded DNA cycle sequencing (Life Technologies) of the P1 genomic clones by using [γ-$#P]dATP end-labelled anti-sense strand oligonucleotides oPC80 (5h-CTCCCTGCGGAACTCG-3h), complementary to nt j6 to j22 of human PC8 [19], and oPC114 (5h-CATGGTCCCTCTTCAGAAG-3h), corresponding to nt k226 to k208 of the human PC8 gene (see Figure 3). To overcome the effects of the GC-rich nature of the 5h-flanking region, DMSO was added to the double-stranded DNA cycle sequencing reactions at a final concentration of 5 mM.…”