Isolation and biochemical characterization of LEAP‐2, a novel blood peptide expressed in the liver

Abstract

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...

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken

“…GAL1-10 Gallinacins (Gal-) 1-10, GAL1A Gal-1α, THP turkey heterophil peptide, TBD turkey β-defensin, BD07 MOUSE mouse β-defensin 7 (Q91V70) intestine, gall bladder and kidneys. These results are consistent with studies of human LEAP-2, which is also expressed in the liver, kidney and colon (Krause et al 2003). Chicken cathelicidin is expressed across a wide variety of tissues, but shows particularly high levels of expression in the bursa, testis and bone marrow, and is the only novel AMP to show expression in the gizzard.…”

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

“…Shike et al (2002) showed that the LEAP-1 gene is expressed strongly in the liver but at lower levels in other tissues of bacterially challenged fish. With the recent discovery of LEAP-2 in mammals (Krause et al, 2003) and birds (Lynn et al, 2003), we have searched the fish EST databases to see if this molecule also exists in bony fish, especially salmonids. We found ESTs with high homology to LEAP-2 in rainbow trout, Atlantic salmon, winter flounder and zebrafish, although the latter was an incomplete sequence.…”

“…Among the cysteine-rich peptides, defensins are the most well known molecules, containing three pairs of disulfidebridges. More recently LEAP (liver-expressed antimicrobial peptide) molecules with four and two pairs of disulfide bonds, named LEAP-1 and LEAP-2 respectively, have been isolated from human blood ultrafiltrate (Krause et al, 2000(Krause et al, , 2003 and the corresponding genes sequenced.…”

“…The LEAP-2 genes reported now include those for human, Rhesus monkey, guinea pig, mouse, cow, pig (Krause et al, 2003) and chicken (Lynn et al, 2003). All the members of the LEAP-2 family have a highly conserved motif containing four cysteine residues which makes it a unique peptide group that does not exhibit similarity with any known peptide family with respect to primary structure, the disulfide motif and expression pattern (Krause et al, 2003).…”

Gene structure and expression characteristics of liver-expressed antimicrobial peptide-2 isoforms in mud loach (Misgurnus mizolepis, Cypriniformes)

“…AMPs disrupt the physical integrity of microbial membranes and also they function as a modulatory effector in the innate immunity of fish (Townes et al 2009;Li et al 2012). Liver-expressed antimicrobial peptide-2 (LEAP-2) is the second blood-derived antimicrobial peptide, which was first identified in human (Krause et al 2003). Similar with LEAP-1 (also termed hepcidin), LEAP-2 is cysteine-rich and predominantly expressed in the liver.…”