Proteinase inhibitors are important negative regulators of proteinase action in vivo. We have succeeded in isolating two previously unknown polypeptides (HF6478 and HF7665) from human blood filtrate that are parts of a larger precursor protein containing two typical Kazaltype serine proteinase inhibitor motifs. The entire precursor protein, as deduced from the nucleotide sequence of the cloned cDNA, exhibits 15 potential inhibitory domains, including the Kazal-type domains, HF6478, HF7665, and 11 additional similar domains. An inhibitory effect of HF7665 on trypsin activity is demonstrated. Because all of the 13 HF6478-and HF7665-related domains share partial homology to the typical Kazal-type domain but lack one of the three conserved disulfide bonds, they may represent a novel type of serine proteinase inhibitor. The gene encoding the multidomain proteinase inhibitor, which we have termed LEKTI, was localized on human chromosome 5q31-32. As shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis, it is expressed in the thymus, vaginal epithelium, Bartholin's glands, oral mucosa, tonsils, and the parathyroid glands. From these results, we assume that LEKTI may play a role in antiinflammatory and/or antimicrobial protection of mucous epithelia.Proteinases are enzymes required for nonspecific processes of digestion and intracellular protein turnover as well as specific proteolytic activation of inactive precursors of many regulatory proteins, such as enzymes and peptide hormones. In addition, they are involved in several processes of extracellular matrix remodeling. Depending on the nature of their reactive center, they are subdivided into the classes of serine, cysteine, aspartate, and metalloproteinases (for review see Ref. 1). To control the action of proteinases in vivo, organisms produce another group of proteins, namely the proteinase inhibitors (for review see Refs. 2-4). Indeed, many pathological effects are due to the non-regulated action of endogenously produced proteinases or such proteinases encoded or synthesized by viruses, bacteria, and parasites (for review see Ref. 5). For instance, a genetically determined fault of the ␣ 1 -proteinase inhibitor may lead to an enhanced proneness to lung emphysema caused by uncontrolled action of leukocyte elastase (6 -8). Thus, proteinase inhibitors represent an important therapeutic tool for a large number of different disorders.Here we report the isolation of two peptides (HF6478 and HF7665) from human blood filtrate (hemofiltrate), which may represent a novel class of proteinase inhibitor. Blood filtrate, a by-product of ultrafiltration of the blood from patients with acute renal failure, is routinely used by us as a source for the systematic as well as random isolation of novel human peptides (9). Due to the cut-off limit of the hemofilters (approximately 20,000 Da), it mainly contains peptides exhibiting a molecular mass below 20,000 Da. Nevertheless, we succeeded in isolating members of many different peptide/protein families suc...

No abstract

The large intestine of breast‐fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin‐treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin‐derived peptide‐I, PRELP‐I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named ''HCC-2.'' The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CK␤8 and the murine chemokines C10, CCF18͞MRP-2, and macrophage inf lammatory protein 1␥, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOHterminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inf lammatory protein 1␣. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.We recently have isolated and characterized a new human CC chemokine, HCC-1 (1), which is structurally similar to macrophage inflammatory protein (MIP)-1␣ (46% amino acid identity) and occurs in high concentrations in human plasma like MIP-1␥ in murine blood. Northern blot analysis revealed that, in contrast to other chemokines, HCC-1 is expressed constitutively at high levels in numerous human tissues. We further showed that HCC-1 interacts with receptors that also recognize MIP-1␣ and RANTES and stimulates the proliferation of CD34 ϩ myeloid progenitor cells in vitro. In the present paper, we report the discovery and characterization of a CC chemokine, HCC-2, that arises from a gene that is arranged in tandem with the gene of HCC-1 on chromosome 17. Mono-as well as bicistronic transcripts of the two genes were detected. Recombinant HCC-2 was expressed and shown to act mainly on monocytes and eosinophils in a similar manner as MIP-1␣. MATERIALS AND METHODSCell Culture. Cell lines (T84, HUH-7) were purchased from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal calf serum.Oligonucleotides. The oligo-deoxyribonucleotides used were synthesized chemically (Perkin-Elmer) and are listed in Table 1.Gene Cloning and Characterization. A human genomic library in phage (Stratagene) was screened as described (2) by using a partial 269-bp, HCC-1-specific cDNA fragment (1) as a probe. Three independent clones were isolated, and the SstI restriction fragments of one of them were subcloned into pBSK ϩ . Both strands of the HCC-1 and HC...