Alexander Krause

LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP‐1 (liver‐expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine‐rich peptides, a 25‐residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP‐1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram‐positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram‐negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose‐dependently exhibited sensitivity upon treatment with synthetic LEAP‐1. The discovery of LEAP‐1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.

Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity.

β‐Defensins are antibiotic peptides involved in host defense at the epithelial surface. Three human β‐defensins (hBDs)‐‐hBD‐1, hBD‐2, and hBD‐3‐‐have been identified so far. We have characterized a new member of the β‐defensin family, hBD‐4, based on screening of genomic sequences and subsequent functional analysis. In contrast to hBD‐1, hBD‐2, and hBD‐3, which are diffusely expressed throughout many organs, hBD‐4 mRNA expression is confined to testis, stomach, uterus, neutrophils, thyroid, lung, and kidney. hBD‐4 expression was up‐regulated by infection with gram‐positive and gram‐negative bacteria in human respiratory epithelial cells, and in response to phorbol 12‐myristate 13‐acetate, but not in response to other inflammatory factors that up‐regulate the expression of hBD‐2 or hBD‐3. Synthetic hBD‐4 exhibits a selective, salt‐sensitive spectrum of antimicrobial activity, and it represents one of the most active antimicrobial peptides against Pseudomonas aeruginosa (minimal inhibitory concentration: 4.1 μg/ml) known to date. This new defensin is chemotactic for human blood monocytes, but it is inactive on neutrophils and eosinophils. These findings demonstrate the existence of a family of β‐defensin genes with different functions against diverse classes of microorganisms, regulated by different stimuli, and specific signal pathways, and confirm the relevance of antimicrobial peptides in host defense.

Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction.

Abstract. Previous studies have shown the implication of β-defensins in host defense of the human body. The human β-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human β-defensin, called human β-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-α (TNF-α), hBD-3 expression is increased particularly after stimulation by interferon-γ. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human β-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

Distribution of new human β-defensin genes clustered on chromosome 20 in functionally different segments of epididymis

Human beta-defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel beta-defensin genes (DEFB25-DEFB29) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of beta-defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation. We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense.

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.