Peter Schulz-Knappe

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.

hBD-1: a novel β-defensin from human plasma

We report the isolation and characterization of a novel peptide with significant sequence homology to β‐defensins from human blood filtrate. The human β‐defensin‐1 (hBD‐1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD‐1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD‐1 shares the nine conserved amino acids characteristic for β‐defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD‐1 is present in nanomolar concentration in human plasma.

Composition of the peptide fraction in human blood plasma: database of circulating human peptides

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Peptidomics technologies for human body fluids

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.

The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa)

Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N‐terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C‐terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3‐kDa peptide in human blood.

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.

GCAP‐II: Isolation and characterization of the circulating form of human uroguanylin

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP‐II). GCAP‐II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C‐terminal amino acids are identical to uroguanylin from human urine. Native and synthetic GCAP‐II activate GC‐C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP‐II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP‐II specific antibodies were used to localize the peptide by immunohistochemistry in entero‐endocrine cells of the colonic mucosa.

Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate

We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification.

Peptide bank generated by large-scale preparation of circulating human peptides.

Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF is obtained in large quantities during treatment of patients suffering from chronic renal failure. We have developed a large-scale method for separating peptides from amounts up to 10,000 1 HF into 300 fractions in a standardized two-step procedure, employing cation-exchange separation, followed by reversed-phase chromatography. These fractions represent a peptide bank containing bioactive, desalted and lyophilized peptides of blood. Screening for and isolation of regulatory human peptides is simplified by using this peptide bank.

Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics.

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.