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
2000-08-01
2003-06-10
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C530S300000, C530S350000, C530S386000, C530S387100, C530S387300, C530S387900, C530S388100, C530S388150, C530S388230, C530S389100, C530S389200, C530S391100, C530S391300, C530S391500, C530S391700, C530S391900
Reexamination Certificate
active
06576445
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel chemokine. More specifically, isolated nucleic acid molecules are provided encoding a human Chemokine Alpha-4, hereinafter referred to as “CK&agr;-4”. CK&agr;-4 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of CK&agr;-4 activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.
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 8-12 kDa in size. 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 subfamnily, 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, over a dozen 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, lysosomal 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 (L-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” subfamily 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 subfamily 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 subfamily act predominantly on mononuclear cells and members of the C—X—C subfamily act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one subfamily show characteristics of the other.
The polypeptide of the present invention has the conserved cysteine residues of the “C—X—C” region, and have amino acid sequence homology to known chemokines.
Clearly, there is a need for factors that regulate the migration of distinct cell types and their roles in dysfunction and disease. There is a need, therefore, for identification and characterization of such factors that regulate the migration of cells, particularly cells of the immune system, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CK&agr;-4 polypeptide having the amino acid sequence shown in
FIG. 1
(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97692 on Aug. 27, 1996. The nucleotide sequence determined by sequencing the deposited CK&agr;-4 clone, which is shown in
FIG. 1
(SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 94 amino acid residues, including an initiation codon at positions 66-68, with a leader sequence of about 18 amino acid residues, and a predicted molecular weight of about 10 kDa. The amino acid sequence of the predicted mature CK&agr;-4 protein is shown in
FIG. 1
, amino acid residues 19-94 (also residues 19-94 in SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CK&agr;-4 polypeptide having the complete amino acid sequence in
FIG. 1
(SEQ ID NO:2); (b) a nucleotide sequence encoding the complete CK&agr;-4 polypeptide having the amino acid sequence shown in
FIG. 1
excepting the N-terminal methionine (i.e., residues 2 to 94 in SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted mature CK&agr;-4 polypeptide having the amino acid sequence at positions 19-94 in
FIG. 1
(SEQ ID NO:2); (d) a nucleotide sequence encoding the CK&agr;-4 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97692; (e) a nucleotide sequence encoding the mature CK&agr;-4 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97692; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e) or (f), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a CK&agr;-4 polypeptide having an amino acid sequence in (a), (b), (c), (d) or (e), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of CK&agr;-4 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated CK&agr;-4 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the CK&agr;-4 polypeptide having the complete 94 amino acid sequence, including the leader sequence shown in
FIG. 1
(SEQ ID NO:2); (b) the amino acid sequence of the CK&agr;-4 polypeptide shown in
FIG. 1
excepting the N-terminal methionine (i.e., residues 2 to 94 shown in SEQ ID NO:2) (c) the amino acid sequence of the predicted mature CK&agr;-4 poly
Olsen Henrik S.
Ruben Steven M.
Zeng Zhi-Zhen
Caputa Anthony C.
Harris Alana M.
Human Genome Sciences Inc.
Human Genome Sciences Inc.
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