LEAP-2

A New Liver-expressed Antimicrobial Peptide 2

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

Krause A., et al. Protein Science (2003), 12:143-152

LEAP-2 immunohistochemistry in liver and kidney tissue

 

Human LEAP-2 precursor cDNA and deduced amino acid sequence. Coding regions are printed in capital letters. The typical secretory signal sequence 1–22 is printed in italics, whereas the positions of introns 1 (198 bp) and 2 (320 bp) of the LEAP-2 gene are marked by vertical lines. Two TATAAA sequences located 31 and 84 bases upstream of the translational start were identified. The putatively mature and antimicrobially active LEAP-2-(38–77) isolated from hemofiltrate and the putative polyadenylation signal are underlined. The position of the primers used for nested 5'-RACE-PCR and for preparative PCR from six different species is indicated.

Alignment ofLEAP-2 from mammalian species. Standard PCR carried out with primers originally designed for the human LEAP-2 gene revealed homologous LEAP-2 forms in rhesus monkey, cow, pig, mouse, and guinea pig. The depicted putative peptide sequences are deduced from the cDNA sequences obtained. Underlined amino acids represent the putative signal peptide sequences as predicted by the SignalP V2.0 program (Nielsen et al. 1997), and the mature LEAP-2 (38–77) form is hyphenated. The nucleotide sequence data reported in this paper have been submitted to the GenBank/EBI Data Bank with accession numbers AJ306405 (Homo sapiens mRNA), AJ409013 (Sus scrofa mRNA), AJ409014 (Bos taurus mRNA), AJ409054 (Cavia porcellus mRNA), AJ409055 (Mus musculus mRNA), AJ409056 (Macaca mulatta genomic DNA), AJ409063 (Mus musculus genomic DNA), AJ409064 (Homo sapiens genomic DNA), and AJ409065 (Homo sapiens alternative promoter sequence).

Tissue distribution of the two main LEAP-2 transcripts. Expression analysis of the LEAP-2 gene was performed by real-time quantitative RT-PCR specific for LEAP-2350 (A) and LEAP-2550 (B) transcripts. Standard curves were constructed using a reference plasmid representing a known number of copies of the target gene as well as the housekeeping gene. Expression levels are therefore depicted as number of copies of LEAP-2 transcript per 108 copies of the housekeeping gene GAPDH. Experiments were performed in duplicate at least twice.

Alternative splicing in the LEAP-2 gene. (A) Standard PCR conducted with the primer combination E1-S and E3-AS (Fig. 2) and 20 ng of DNase-treated cDNA. LEAP-2350 is mainly expressed in liver, kidney, and colon, whereas LEAP-2550 is the main transcript in lung, trachea, and heart. PCR products at 650 bp, 720 bp, and 870 bp represent splicing variants of LEAP-2, and the band at 480 bp could be identified as a PCR artefact. (MW = marker, 100 bp DNA ladder, Life Technologies). (B) Standard PCR performed with the primers PROM-S (5'-GGTGCA GATTAGGGTGACAGTCCATC-3'), which is located 565 bp upstream of the transcriptional start depicted in Figure 2, and E3-AS. Lung, heart, and trachea exhibit the same pattern of bands, including the 565 bp shift in size caused by the upstream primer PROM-S. The main transcript identified in liver, kidney, and colon, however, changes from the completely spliced LEAP-2350 to the intron 1-retaining LEAP-2 variant. All bands obtained were characterized by DNA sequencing.

Northern Blot analysis. Human MTN Blots I+II (Clontech, A+B) were hybridized under high-stringency conditions with a 32P-labeled LEAP-2-specific cDNA fragment. A transcript size of 0.7 kb identified in liver, kidney, and small intestine represents the complete LEAP-2 cDNA including a poly(A)-tail of 200 bp. The signal at 2.0 kb corresponds to the LEAP-2 form coded by the distal promoter, which could also be identified in 5'-RACE-PCR, whereas the bands at 4.2 kb and 8.0 kb might either represent additional alternative promoter variants or homologous proteins related to LEAP-2.

Antimicrobial activity of LEAP-2-(38–77) and LEAP-2-(44–77). Colony-forming unit assay of synthetic LEAP-2-(38–77) (solid triangles) and LEAP-2-(44–77) (open circles), the two main peptide forms isolated from hemofiltrate, against S. cerevisiae ATCC9763. Incubation without peptide represents 100% CFU. Synthetic and native LEAP-2-(44–77) led to similar results. The bars indicate the minimum and maximum value of the triplicates used in this representative assay. The inset depicts the dose-dependent effect of LEAP-2-(38–77) against S. cerevisiae in a radial diffusion assay. LEAP-2-(44–77) showed no effect in this sensitive antimicrobial assay. The antimicrobially active casocidin-I (11 µg/well) served as a positive control ( Zucht et al. 1998). One inhibition unit (1 IU) corresponds to 0.1 mm diameter of growth inhibition zone, and the error bars represent the S.D. calculated from three experiments performed.