Human Defensin DEF-X

Details Human Defensin DEF-X Human Defensin DEF-X

2000-02-25

2001-12-11

Gambel, Phillip (Department: 1644)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S001000, C514S002600, C514S885000, C514S886000, C514S887000, C530S300000, C530S324000

Reexamination Certificate

active

06329340

ABSTRACT:

This application claims priority, under 35 U.S.C. § 371, to PCT/FR98/01864.
The present invention relates to a novel human polypeptide defensin Def-X, homologous to HNP-4, its genomic DNA and CDNA.
The invention also relates to cloning and expression vectors, and cells transformed with said vectors. The subject of the invention is also the use of said polypeptides as antibiotic, cytotoxic, repair and endocrine regulatory agent and as pesticide as well as cosmetic or pharmaceutical compositions for the treatment of microbial infections, in particular bacterial, fungal and viral infections, or parasitic infections, cancers, of inflammation and of immune deficiency. Finally, the invention comprises diagnostic methods and kits for the determination of a microbial or parasitic infection and of an inflammation, or for screening for predisposition to immune deficiencies or cancer diseases.
Antimicrobial substances are key elements in the defence of multicellular organisms. Among these substances, there are both simple inorganic compounds (hydrogen peroxide, hypochlorous acid, nitric oxide) and complex proteins and peptides. They are present at the first lines of defence, at the surface of the mucous membranes of various organs, in particular in the epithelial cells of the intestine and of the lungs, depending on the species, as well as in the microbicidal organs of phagocytic cells of hematopoietic origin, where they were first identified. Their synthesis de novo or their release from storage sites—organelles of the lysosome or cytoplasmic granule type which are capable of storing them in an inactive or latent form—can be induced rapidly, which makes them particularly important in the early phases of resistance to infections (Martin et al., 1995).
The antimicrobial proteins of less than one hundred aminoacids in size are arbitrarily called antimicrobial peptides. Several families of antimicrobial peptides have been identified, which differ in the presence within them of disulfide bridges, in their aminoacid composition, their structural conformation and in their activity spectrum. The antimicrobial peptides comprising six conserved cysteines form the defensin family. This family is composed of antimicrobial peptides which are present in numerous species, which are abundant and which are about 3-4 kDa (Ganz and Lehrer, 1994). These peptides are formed of 30 to 40 aminoacids, of which six invariant cysteins which form three intramolecular disulfide linkages. They have complex conformation, are amphipathic, rich in beta antiparallel sheets but lack alpha helices (Lehrer and Ganz, 1992). The antimicrobial action of defensins is thought to result from their insertion into the membranes of the target cells, allowing the formation of voltage-dependent channels. White et al. (1995) describe the possible mechanisms of membrane insertion and of formation of multimeric pores by the defensins, which allow the permeabilization of the membranes of the target cells, for example microbial or tumor cells. The crystallographic structure of human neutrophil defensin HNP-3 (see below) has been determined, and a specific mechanism of dimerization of the human neutrophil defensins is in addition suggested. Increased knowledge of this family of peptides and comparison of their sequences and activity spectra will make it possible to better understand these mechanisms and their specificities, as well as the aminoacid residues more particularly involved in these phenomena.
The defensins are divided into three familities of peptides which are structurally different: the “conventional” defensins, the beta-defensins and the insect defensins. These families exhibit differences as regards the position of and the distance between the conserved cysteine residues, as well as those of other conserved aminoacids (proline, glycine) (Ganz and Lehrer, 1995).
Human defensins, of the conventional type, come essentially from two sources. They were first identified by peptide purification from neutrophil extracts. Four defensins have thus been isolated: “human neutrophil peptides” HNP-1, HNP-2, HNP-3, and HNP-4. The first three are different products of the same gene (Ganz et Lehrer, 1995). These three peptides represent 99% of the defensin content of the neutrophils, whereas HNP-4 is also present therein, but at concentrations which are 100 times lower. More recently, two human enteric defensins, HD-5 and HD-6, were characterized in the small intestine and more precisely in the Paneth cells (Bevins et al., 1996). While 16 enteric defensin genes have been identified in mice, only these two homologs have been identified in humans (Mallow et al., 1996).
Defensins have an antimicrobial action on a broad spectrum of microorganismes in vitro (Martin et al., 1995). This activity spectrum, which is particularly broad, comprises bacteria, Gram-positive and Gram-negative bacteria, several fungi, mycobacteria, parasites including spirochetes and several enveloped viruses including the HSV and HIV viruses. They are also cytotoxic for several categories of normal and malignant cells, including cells resistant to TNF-alpha and to the cytolytic NK factor (Kagan et al., 1994). The large quantity of targets of the defensins and their abundance in blood cells specialized in the immune defence, as well as the dramatic increase in their concentration during severe infections, suggest that these molecules could play an important role in the natural immunity to infections and to cancers. In particular, the increase in the transcription of the defensin genes and the release of cytoplasmic granules containing presynthesized defensins in response to stimuli, contributes to the local antimicrobial response, it being possible for the defensins to participate in the inflammatory reaction, in the repair processes and in endocrine regulation during infection. The hematopoietic defensins could contribute to the phenomenon of lysis of cancer cells, a phenomenon which is mediated by the neutrophils during the antibody-dependent immune response. The precise physiological role of the enteric defensins is not clearly established. They could stem the proliferation of the intraluminal flora or prevent the translocation of bacteria across the intestinal mucosa (Mallow et al., 1996). The abundance of the defensin mRNA in the Paneth cells reinforces the hypothesis that these epithelial cells could play a key role in the immune defence in the intestine. It has moreover been shown that their expression scheme coincides with the appearance of the Paneth cells during embryogenesis. Mallow et al. (1996) have suggested that low levels of expression of enteric defensins in the fetus could be the evidence of an immaturity of a local defence, which would predispose children born prematurely to infections due to intestinal microorganisms.
A defensin concentration corresponding to 10% of the normal level is observed in patients suffering from “specific granule deficiency”, a rare disease of the development of the granulocytes. The affected subjects suffer from frequent infections caused by common bacteria (Ganz and Lehrer, 1995).
Biochemically modified defensins are potential prophylactic and therapeutic agents against infections (Ganz and Lehrer, 1995). Research relating to these antimicrobial peptides or other molecules participating in the natural immunity have gained special importance since phenomena of resistance of microorgnisms to traditional antibiotics started to develop (Bevins et al., 1996).
The primary structure of defensins, in particular of human defensins, has been the subject of recent studies (White et al., 1995; Mallow et al., 1996). The conventional defensins comprise 29 to 35 aminoacids, but are derived from precursors—preproteins—comprising 90 to 100 aminoacids. The proteolytic maturation of the human neutrophil defensins to mature peptides is coupled with their despatch to the granulocytes; the function of the propeptide would include the inactivation of the precursor form of defensin and a support for the acquisition of the active conformation of the

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