The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene-encoded, cysteine-rich, and cationic peptide that we termed liver-expressed antimicrobial peptide 2 (LEAP-2). We identified several circulating forms of LEAP-2 differing in their amino-terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1-3 and 2-4 positions. Molecular cloning of the cDNA showed that LEAP-2 is synthesized as a 77-residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP-2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue-specifically expressed. The largest native LEAP-2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin-like endoprotease. In contrast to smaller LEAP-2 variants, this peptide exhibited dose-dependent antimicrobial activity against selected microbial model organisms. LEAP-2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.Keywords: Alternative splicing; antimicrobial activity; disulfide bonds; hemofiltrate; liver; peptide; secretion As a consequence of the efforts to sequence and assemble the human genome (Lander et al. 2001;Venter et al. 2001), the systematic analysis of peptides and proteins as the functional gene products produced by a given cell population or tissue under defined conditions is considered to be the next milestone in molecular biology. The estimated number of genes is unexpectedly low, and the number of biologically active peptides and proteins cannot be deduced from these data because of events such as alternative splicing of mRNA precursors, usage of alternative gene promoters, pseudogenes, and alternatively processed proteins. The number of proteins is therefore estimated to be two to three orders of magnitude higher than the number of ∼40,000 genes annotated in the human genome (Harrison et al. 2002;Rappsilber and Mann 2002). Therefore, the mass-spectrometric identification of a gene product in proteomics or, even better, the isolation of novel proteins and peptides followed by a struc-5 Reprint requests to: Knut Adermann, IPF PharmaCeuticals GmbH, Hannover, Germany; e-mail: knut.adermann@ipf-pharmaceuticals.de; fax: 49 (0) 511 5466 132.Abbreviations: CFU, colony-forming unit; ESIMS, electrospray ionization mass spectrometry; LEAP, liver...
Members of the Ly-60uPAR protein family share one or several repeat units of the Ly-60uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-60uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1~secreted mammalian Ly-60uPAR related protein 1! from blood and urine peptide libraries. SLURP-1 is encoded by the ARS~component B!-810s locus, and appears to be the first mammalian member of the Ly-60uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-60uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.
We identified two regulatory elements in the upstream region of the Drosophila Sgs-3 gene which are both able to bind the ecdysone receptor (EcR/USP) and the product of the fork head gene. Interestingly, only one of the EcR/USP binding sites is able to recognize in vitro-translated EcR/USP, which provides evidence for the existence of different receptor forms having different DNA binding specificities. Deletions of the elements lead to a reduced accumulation of Sgs-3 mRNA without altering the temporal expression profile of the gene. The data are consistent with the hypothesis that the ecdysone receptor directly contributes to the transcriptional activation of Sgs-3 by binding to at least one of the two elements. Since also the Sgs-4 gene is controlled by a functional EcR/USP binding site, a direct participation of EcR/USP in the formation of regulatory complexes may be of general importance for the hormonal control of Sgs genes.
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