IgE-dependent histamine-releasing factor-binding peptides

Details IgE-dependent histamine-releasing factor-binding peptides IgE-dependent histamine-releasing factor-binding peptides

2001-06-01

2004-03-23

Kunz, Gary (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C424S084000, C514S002600

Reexamination Certificate

active

06710165

ABSTRACT:

TECHNICAL FIELD
The present invention relates to IgE-dependent histamine-releasing factor (hereinafter, abbreviated as “HRF”) receptor, HRF-binding peptides and nucleic acids encoding the same, and uses thereof. More specifically, the present invention relates to novel receptors against HRF causing allergic diseases such as asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, allergies due to foods, drugs, pollen, insects, etc., hay fever, cold urticaria or atopic dermatitis, HRF-binding peptides and nucleic acid encoding the same, and uses thereof in the medicinal area.
BACKGROUND ART
Allergies are known as being caused by inheritable hypersensitive formation of IgE in response to allergens, or disruption of balance between IL-4 (Interleukin-4) increasing IgE secretion and interferon decreasing IgE secretion. Upon the exposure to allergens, an immediate reaction occurs and various cells associated with inflammation are gathered, and after several hours, late-phase reaction (hereinafter, abbreviated as “LPR”) occurs by histamine and other cytokines secreted from basophils, eosinophils and lymphocytes. In LPR, histamine is secreted from basophils, but allergens, which have initiated the reaction, do not exist any longer. Further, LPR is developed in only about half of patients suffered from allergies. Therefore, what causes histamine secretion from basophils and what causes development into LPR have been issues of great interest. To the present time, cytokines such as MCP-3, MCP-1 or RANTES were known as secreting histamine. But, it was found that in IgE-dependent LPR, only HRF can induce histamine secretion from basophils (MacDonald, et al., 1995), the mechanism of which has never been known.
HRF, which is a ubiquitous cytoplasmic protein, is a known protein consisting of 172 amino acids (Bohm, et al., 1989). 45 Amino acids at its C-terminal form basic domain. Because such domain has about 46% homology with MAP-1B, microtubule-associated protein, it was assumed that HRF is also microtubule-associated protein. Gachet, et al. (1997) observed that HRF is distributed consistently with the cytoskeletal network to some extent by using confocal microscope, which suggests that HRF binds to the cytoskeleton. Meanwhile, Sanchez, et al. (1997) published that HRF, even though it does not fall within general Ca
2+
-binding protein family, binds to Ca
2+
and further, identified that yeast cells can survive with the deletion of HRF genes in
Saccharomyces cerevisiae
. These suggest that HRF falls within the gene family having redundant pathway. MacDonald, et al. (1995) also found HRF, which is an intracellular protein, in the outside of cells. Further, it was known that HRF present in the outside of cells stimulates IgE-sensitized basophils to release histamine, but an accurate interaction between IgE and HRF has not been identified (Schroeder, et al., 1996). Schroeder, et al. (1997) observed that HRF can augment the anti-IgE-induced histamine release from all basophils, regardless of the IgE type, and thus suggested that HRF exerts its function by binding to cell membrane receptors, not by binding with IgE. Accordingly, the followings have been important issues, i.e. how HRF is secreted to the outside of cells and how it stimulates IgE-sensitized basophils to release histamine. Since HRF, a hydrophilic and intracellular protein, is detected in LPR allergy patients plasma at a large amount, it was assumed to be secreted to the outside of cells by apoptosis or other mechanisms and to release histamine via HRF receptors present in basophil membrane. In addition, because this HRF exists in most of tissues, it is assumed to function in tissue cells other than in inflammatory cells. But, its functions in other tissues than inflammatory cells, particularly in cerebral tissue or nerve cells, have never been reported. Recently, HRF was found during the analysis of proteins present in human brain using 2-D gel electrophoresis and proteomics (Langen, et al., 1999). Subsequently, it was also reported that HRF protein is decreased in the brain of patients died of Down's syndrome or Alzheimer's disease (Kim, et al., 2001).
On the other hand, (Na,K)ATPase, which involves in the formation of resting membrane potential and in the balanced regulation of osmosis within cells, is also present in nerve cells, particularly nerve end or synaptosomal membranes, at a high concentration and plays an important role in neuroactivity. It was reported that in case of inhibition or loss of (Na,K)ATPase activity in nerve cell membrane, various neuropathological changes or apoptosis occurs (Lees, 1991). This is also related to the report that the intracellular ATP essential for (Na,K)ATPase activity is rapidly exhausted in cerebral ischemia or anoxia state (Martin, et al., 1994; Santos, et al., 1996). Therefore, it is believed that this enzyme activity is also inhibited in such cerebral disease states. Moreover, it was confirmed that in rat brain tissue slices, synaptosomes and in vitro culture system, in case (Na,K)ATPase activity is inhibited, neurotransmitters release is increased. From other in vivo and in vitro studies, it was suggested that neurotransmitters release is increased in ischemia or anoxia-like conditions and the resulting activation of postsynaptic cell membrane receptors is an important procedure in nerve injury (Choi, 1990; Martin, et al., 1994).
Cerebral (Na,K)ATPase activity is regulated not only by neurotransmitters such as dopamine, serotonin, norepinephrine, glutamate, etc. but also by endogenous substances such as insulin, nitric oxide (NO), etc. An endogenous (Na,K)ATPase inhibitor named “brain ouabain”, which is structurally similar to ouabain, glycoside extracted from plants, was identified (Budzikowski, et al., 1998). But, Rodriguez, et al. (1992) reported that there exists an endogenous ouabain-like factor specifically inhibiting (Na,K)ATPase activity in soluble brain fractions and having the different structure and properties from ouabain. They also reported that it blocks high affinity
3
H-ouabain binding to induce neurotransmitters release, and involves in (Na,K)ATPase activity regulation by neurotransmitters as well. Recently, that substance was named endobain E (Vatta, et al., 1999), bur has not yet been identified.
DISCLOSURE OF THE INVENTION
Surprisingly, the present inventors found that HRF, even though it is a hydrophilic protein, can transit the cell membrane and HRF receptor corresponds to a third cytoplasmic domain (CD3) of (Na,K)ATPase by yeast two-hybrid assay. In addition, the inventors first identified an accurate mechanism by which extracellularly secreted HRF stimulates histamine release within basophils.
Further, on the basis of the results as described above, the inventors anticipated that any allergic diseases can be effectively prevented or treated by blocking HRF introduction into the cells and/or HRF binding with (Na,K)ATPase to inhibit histamine release. Therefore, they have performed extensive studies on peptides binding to HRF by screening 12 mer and 7 mer phage display libraries and as a result, obtained peptides of the specific sequences which can inhibit histamine secretion at a remarkably high rate and thus, completed the present invention.
Accordingly, an object of the present invention is to provide novel HRF receptors, peptide binding to HRF and uses thereof.
A first aspect of this invention relates to a rat HRF receptor having the amino acid sequence selected from SEQ ID No. 1, 2 or 3.
A second aspect thereof relates to a human HRF receptor having the amino acid sequence selected from SEQ ID No. 4, 5 or 6.
A third aspect thereof relates to a HRF receptor having the sequence homology of 85% or more with any one of the above amino acid sequences.
The HRF receptor may be a large cytoplasmic loop [CD (cytoplasmic domain) 3] of (Na,K)ATPase &agr;1, &agr;2 or &agr;3 subunit.
A fourth aspect of this invention relates to a nucleic acid encoding any one of the above HRF receptors. The nucleic acid may have the nucleotide

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