Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-11-18
2002-05-28
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S071200, C435S471000, C435S320100, C435S325000, C435S252300, C435S254110, C536S023100, C536S023500, C530S351000, C424S085100
Reexamination Certificate
active
06395514
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel human gene encoding a polypeptide which is a member of the chemokine family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Chemokine Alpha-5, hereinafter referred to as “CK&agr;-5”. CK&agr;-5 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the immune system, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of CK&agr;-5 activity.
BACKGROUND OF THE INVENTION
The ability to control the migration and “trafficking” of various cell types is controlled by a subset of factors, or proteins, among which chemokines are an example.
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related chemotactic cytokines. These molecules are usually 8-10 kd in size, however, larger membrane bound chemokine polypeptides have been identified. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the “C—X—C” subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the “C—C” subfamily. Thus far, at least sixteen different members of this family have been identified in humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysogomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
Members of the “C—C” branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophages which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation. However, members of one branch may exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches.
While members of the C—C branch act predominantly on mononuclear cells and members of the C—X—C branch act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one family show characteristics of the other.
The polypeptide of the present invention has the conserved cysteine residues of the “C—X—C” region, and has amino acid sequence homology to known chemokines.
Thus, there is a need for polypeptides that function as regulators of the migration of distinct cell types and of their roles in dysfunction and disease, since disturbances of such regulation may be involved in disorders relating to hemostasis, angiogenesis, tumor metastisis, cellular migration and ovulation, as well as neurogenesis. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating or correcting such disorders.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the CK&agr;-5 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by a cDNA clone deposited as plasmid DNA as ATCC® Deposit Number 209231 on Aug. 29, 1997. The ATCC® is located at 10801 University Boulevard, Manassas, Va. 20110-2209. The nucleotide sequence determined by sequencing the deposited CK&agr;-5 clone, which is shown in
FIG. 1
(SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 254 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 542-544. Nucleic acid molecules of the invention include those encoding the complete amino acid sequence excepting the N-terminal methionine shown in SEQ ID NO:2, or the complete amino acid sequence excepting the N-terminal methionine encoded by a cDNA clone in ATCC® Deposit Number 209231, which molecules also can encode additional amino acids fused to the N-terminus of the CK&agr;-5 amino acid sequence.
The polypeptide of the present invention has amino acid sequence homology to known chemokines, including the conserved C—X—C cysteine pattern characteristic of the alpha subfamily of chemokines beginning with the first cysteine from the amino terminus in SEQ ID NO:2.
CK&agr;-5 also lacks the ELR motif found in some alpha chemokines immediately preceding the first cysteine residue, which is known to be required for the neutrophil and endothelial cell chemotactic activity as well as the angiogenic activity of IL-8.
The encoded polypeptide has a predicted leader sequence of 27 amino acids underlined in
FIG. 1
; and the amino acid sequence of the predicted mature CK&agr;-5 protein is also shown in
FIG. 1
as amino acid residues 29-254 and as residues 28-254 in SEQ ID NO:2. Further the polypeptide is predict domain having the sequence from residues 28 to 205 in
FIG. 1
(28 to 205 in SEQ ID NO:2), a transmembrane domain having a sequence from residue 206 to 227 in
FIG. 1
(206 to 227 in SEQ ID NO:2), and an intracellular domain having a sequence from residue 228 to 254 in
FIG. 1
(228 to 254 in FIG.
1
(201 to 227 in SEQ ID NO:
2).
Thus, one aspect of the invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CK&agr;-5 polypeptide having the complete amino acid sequence in
FIG. 1
(SEQ ID NO:2); (b) a nucleotide sequence encoding the CK&agr;-5 polypeptide having the complete amino acid sequence in (i.e., positions 2 to 254 of SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted mature CK&agr;-5 polypeptide having the amino acid sequence at positions 28-254 in
FIG. 1
(28-254 in SEQ ID NO:2); (d) a nucleotide sequence encoding the predicted extracellular domain of the CK&agr;-5 polypeptide having the amino acid sequence at positions 28-205 in
FIG. 1
(28-205 in SEQ ID NO:2); (e) a nucleotide sequence encoding the CK&agr;-5 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC® Deposit No. 209231; (f) a nucleotide sequence encoding the CK&agr;-5 polypeptide having the complete amino acid sequence excepting the N-terminal methionine encoded by the cDNA clone contained in ATCC® Deposit No. 209231; (g) a nuc
Li Yi
Ni Jian
Rosen Craig A.
Ruben Steven M.
Wei Ying-Fei
Human Genome Sciences Inc.
Human Genome Sciences Inc.
Mertz Prema
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