GENOMIC ORGANIZATION AND ALTERNATIVE SPLICING OF HUMAN PACE4 (SPC4), KEXIN-LIKE PROCESSING ENDOPROTEASE

PACE4 (paired basic amino acid cleaving enzyme) is a member of a famil y of the mammalian kexin-like proprotein convertases containing a subt ilisin-like catalytic domain. Previously we reported seven isoform mRN As of PACE4 that vary in size and 3'-coding sequence [A. Tsuji et al. (1994) Biochem. Biophys. Res. Commun. 200, 943-950; K. Mori et al. (19 97) J. Biochem. 121, 941-948]. To determine the origin of these isofor ms, the entire human PACE4 gene has been isolated as a set of overlapp ing genomic DNA fragments, and analyzed by restriction enzyme digestio n and nucleotide sequence determination. The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in size from 39 to 1,422 base pairs. Human PACE4 gene is the largest kexin-like pr oprotein convertase gene reported to date. The most striking feature o f its genomic structure is the size of the introns and the number of e xons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very si milar to that reported for other kexin-like protease genes. The struct ural analysis of PACE4 genomic DNA indicates that multiple PACE4 trans cripts are produced as a consequence of alternative RNA splicing event s, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites. A major transcriptional s tart site was detected 314 bp upstream from the ATG translational star t site by primer extension analysis. Sequence analysis of the 5'-flank ing region revealed that PACE4 gene lacks TATA and CCAAT boxes in the proximal upstream region of the start site, although potential binding sites for several transcription factors including SP1, AP1, AP2, PEA3 , Ets-1, GHF (growth hormone factor)-1, CREB (cyclic AMP response elem ent binding protein), and basic helix-loop-helix proteins, were presen t. An unusual. sequence of six tandem repeats of a nonadecamer (GGCCTG GGGGTTCACCTGC) containing an E box is found in the 5'-flanking region. These results suggest that PACE4 is not a constitutive gene product a nd its expression is regulated by various transcription factors.