Physiological function of proprotein convertase PACE4

PACE4(配对碱性氨基酸裂解酶)是哺乳动物Kexin样原蛋白转换酶家族中的一员，含有枯草杆菌样催化区。为了确定这些异构体的来源，我们分离了整个人类PACE4基因，作为一组重叠的基因组DNA片段，并通过限制性内切酶和核苷酸序列测定进行了分析。人类PACE4基因全长至少250 kb，分布在25个外显子上，e从39到1,422个碱基对不等。人PACE4基因是迄今为止报道的最大的类可信原蛋白转换酶基因。其基因组结构最显著的特征是内含子的大小和外显子的数量，尽管该家族中保守的信号肽、前肽和催化域的总体结构与已报道的其他可信蛋白水解酶基因非常相似。对PACE4基因组DNA的结构分析表明，由于不同的RNA剪接事件，包括外显子跳过，以及内部5‘-剪接供体和聚腺苷酸化位点的使用不同，PACE4产生了多个转录本。通过引物延伸分析，在ATG翻译起始点的上游314bp处检测到一个主要的转录起始点。对PACE4基因5‘侧翼区的序列分析表明，PACE4基因起始区上游附近没有TATA和CCAAT盒，但存在SP1、AP1、AP2、PEA3、ETS-1、GHF(生长激素因子)-1、CREB(环AMP反应元件结合蛋白)和碱性螺旋-环-螺旋蛋白等转录因子的潜在结合位点。在5-侧翼区发现了一个含有E盒的非十八聚体(GGCCTGGGGGGGTCACCTGC)的6个不寻常的串联重复序列。这些结果表明，PACE4不是一个结构性基因产物，它的表达受多种转录因子的调控。

PACE4 (paired basic amino acid cleaving enzyme) is a member of a family of the mammalian kexin-like proprotein convertases containing a subtilisin-like catalytic domain. To determine the origin of these isoforms, the entire human PACE4 gene Las been isolated as a set of overlapping genomic DNA fragments, and analyzed by restriction enzyme digestion and nucleotide sequence determination. The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in e from 39 to 1,422 base pairs. Human PACE4 gene is the largest kexin-like proprotein convertase gene reported to date. The most striking feature of its genomic structure is the size of the introns and the number of exons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very similar to that reported for other kexin-like protease genes. The structural analysis of PACE4 genomic DNA indicates that multiple PACE4 transcripts are produced as a consequence of alternative RNA splicing events, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites. A major transcriptional start site was detected 314 bp upstream from the ATG translational start site by primer extension analysis. Sequence analysis of the 5'-flanking 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 element binding protein), and basic helix-loop-helix proteins, were present. An unusual sequence of six tandem repeats of a nonadecamer (GGCCTGGGGGTTCACCTGC) containing an E box is found in the 5-flanking region. These results suggest that PACE4 is not a constitutive gene product and its expression is regulated by various transcription factors.