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
1998-09-03
2001-09-11
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023500, C536S024310, C435S320100, C435S252300, C435S325000
Reexamination Certificate
active
06287808
ABSTRACT:
BACKGROUND OF THE INVENTION
Members of the tumor necrosis factor receptor (TNFR) superfamily regulate a diverse range of cellular processes including cell proliferation, programmed cell death and immune responses. Characteristically, these receptors are transmembrane (type 1) glycoproteins having cysteine-rich subdomains in their extracellular, ligand binding domain (Gruss (1996)
Int. J. Clin. Lab. Res.
26:143-159).
A recently identified member of the TNFR superfamily is the herpesvirus entry mediator (HVEM) (Montgomery et al. (1996)
Cell
87:427-436). HVEM mediates the entry of many strains of herpes simplex virus (HSV) into cells. Studies have revealed that HSV initiates infection by binding cell surface glycosaminoglycans. To actually enter the cell, the virus requires mediator activity, which is provided by HVEM. HVEM interacts with the virus by binding to the envelope glycoprotein D (gD) and triggering membrane fusion (Whitbeck et al. (1997)
J. Virol.
71:6083-6093; Montgomery et al., supra).
To date, two ligands of HVEM have been identified, LIGHT and Lymphotoxin &agr; (LT&agr;) (Mauri et al. (1998) Immunity, 8:21-30). LIGHT is a novel cytokine and is termed LIGHT because it shows homology to Lymphotoxins, exhibits Inducible expression and competes with HSV Glycoprotein D for HVEM, a receptor expressed by T lymphocytes. The second identified ligand of HVEM, LT&agr;, is expressed exclusively by T-cells, has 30% sequence identity to TNF, and competes with TNF for binding to the TNF1 receptor. The biological effects exerted by LT&agr; are similar to those of TNF. However, unlike TNF, LT&agr; usually acts as a local paracrine factor. LT&agr; has been shown to be a potent activator of neutrophils. Accordingly, it is thought to be a regulator of acute phase inflammatory reactions. In addition, LT&agr; facilitates leukocyte extravasation by increasing leukocyte adhesion and cytokine production.
Recent evidence suggests that HVEM may also play a role in regulating immune responses. Studies have revealed that HVEM can bind to several TNF receptor-associated factors (TRAFs). TRAFs activate stress activated protein kinase-1/c-Jun N-terminal kinase (JNK/SAPK), as well as the transcription factors, Nuclear Factor-KAPPA B (NF-kB), and transcription factor activator protein-1 (AP-1). These transcription factors in turn control the expression of multiple immune, inflammatory, and acute phase genes (Marsters et al. (1997)
J. Biol. Chem.
272:14029-14032).
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of two cDNA molecules which encode soluble forms of the membrane-bound herpesvirus entry mediator (mHVEM), a member of the TNFR superfamily. The cDNA (SEQ ID NO:1) for the first soluble form, soluble herpesvirus entry mediator-1 (sHVEM1), and the cDNA (SEQ ID NO:17) for the second soluble form, soluble herpesvirus entry mediator-2 (sHVEM2), are described below.
The sHVEM1 cDNA (SEQ ID NO:1) has a 579 nucleotide open reading frame (nucleotides 297-875 of SEQ ID NO:1; SEQ ID NO:3) which encodes a 193 amino acid protein (SEQ ID NO:2). This protein includes a predicted signal sequence of about 38 amino acids (from amino acid 1 to about amino acid 38 of SEQ ID NO:2; SEQ ID NO:5; encoded by nucleotide 297 to 410 of SEQ ID NO:1; SEQ ID NO:6). sHVEM1 has a predicted mature protein length of about 155 amino acids (from about amino acid 39 to amino acid 193 of SEQ ID NO:2; SEQ ID NO:
4).
sHVEM1 protein possesses three of the four cysteine-rich repeats/domains characteristic of members of the TNFR family. The first cysteine rich domain is 35 amino acids long (amino acid 41 to about amino acid 75 of SEQ ID NO:2; SEQ ID NO:7). The second cysteine rich domain is 44 amino acids long (amino acid 77 to about amino acid 120 of SEQ ID NO:2; SEQ ID NO:8). The third cysteine rich domain is 45 amino acids long (amino acid 121 to about amino acid 165 of SEQ ID NO:2; SEQ ID NO:9). sHVEM1 is predicted to have two potential N-linked glycosylation sites at amino acid 110 of SEQ ID NO:2 and at amino acid 173 of SEQ ID NO:2.
The sHVEM2 cDNA (SEQ ID NO:17) has a 591 nucleotide open reading frame (nucleotides 107-697 of SEQ ID NO:17; SEQ ID NO:19) which encodes a 197 amino acid protein (SEQ ID NO:18). This protein includes a predicted signal sequence of about 38 amino acids (from amino acid 1 to about amino acid 38 of SEQ ID NO:18; SEQ ID NO:21; encoded by nucleotide 107 to 220 of SEQ ID NO:17; SEQ ID NO:22). sHVEM2 has a predicted mature protein length of about 159 amino acids (from about amino acid 39 to amino acid 197 of SEQ ID NO:18; SEQ ID NO:20). sHVEM2 protein possesses three of the four cysteine-rich repeats/domains characteristic of members of the TNFR family. The first cysteine rich domain is 35 amino acids long (amino acid 41 to about amino acid 75 of SEQ ID NO:18; SEQ ID NO:23). The second cysteine rich domain is 44 amino acids long (amino acid 77 to about amino acid 120 of SEQ ID NO:18; SEQ ID NO:24). The third cysteine rich domain is 45 amino acids long (amino acid 121 to about amino acid 165 of SEQ ID NO:18; SEQ ID NO:25). sHVEM2 is predicted to have two potential N-linked glycosylation sites at amino acid 110 of SEQ ID NO:2 and at amino acid 173 of SEQ ID NO:2.
sHVEM2 is 5 amino acids longer than sHVEM1. Overall, sHVEM1 and sHVEM2 share a high degree of sequence identity, exhibiting 76.6% sequence identity at the nucleotide level and 93.9% sequence identity at the amino acid level. The two proteins are identical from amino acid 1 to amino acid 184. It is only at the very C-terminal end of each protein, from amino acid 185 onwards, that their respective sequences differ. sHVEM1 has 9 C-terminal amino acids (amino acid 185-194 of SEQ ID NO:2) that are distinct from the 13 amino acids at the C-terminal end of sHVEM2 (amino acid 185-197 of SEQ ID NO:18).
Nucleotide sequence and amino acid sequence analysis also revealed that the sHVEM1 and sHVEM2 have particularly high sequence identity with membrane-bound herpesvirus entry mediator (mHVEM), a member of the TNF receptor (TNFR) superfamily. For example, sHVEM1 displays 85.1% nucleotide sequence identity and 65% amino acid sequence identity with mHVEM. However, the sHVEM1 and sHVEM2 sequences differ from mHVEM sequence in two important ways. First, sHVEM1 and sHVEM lack the C-terminal end of mHVEM (amino acids 184-283 of SEQ ID NO:13) which contains the transmembrane domain of mHVEM (amino acids 203-225 of SEQ ID NO:13). The absence of a transmembrane domain in sHVEM1 and sHVEM2 suggests that sHVEM1 and sHVEM2 act as soluble receptors. Second, sHVEM1 and sHVEM2 have additional amino acids at their C-terminal ends that are not found at the C-terminal end of mHVEM, e.g., sHVEM1 contains end an additional 10 amino acids at its C-terminal (amino acid 184-193 of SEQ ID NO:2) and sHVEM2 contains an additional 14 amino acids at its C-terminal end (amino acid 184-197). Moreover, these amino acid sequences do not appear to have significant sequence identity with any other known protein.
mHVEM was first identified by its ability to mediate entry of herpes-simplex virus (HSV) into cells (Montogomery et al., supra). Two ligands for mHVEM have been identified, LIGHT (also called TANGO-69, see U.S. Ser. No. 09/146,951, filed Sep. 3, 1998, hereby incorporated by reference) and LT&agr; (Mauri et al., supra). The mechanism by which mHVEM mediates entry of HSV into cells and the role of LIGHT/TANGO-69 and/or LT&agr; in this mechanism is not understood at present. However, it is known that LIGHT/TANGO-69 can compete with HSV for binding to mHVEM (Mauri et al., supra).
As used herein, the term TANGO-69-receptor refers to the sHVEM1 gene, sHVEM2 gene, sHVEM1 and SHVEM2 genes, sHVEM1 protein, sHVEM2 protein, sHVEM1 and sHVEM2 proteins including any and all gene products encoded by the cDNA of SEQ ID NO:1 and SEQ ID NO:17, as described above.
The TANGO-69-receptor is classified as a member of the TNFR superfamily and is predicted to be the soluble form of mHVEM. Soluble forms for most TNFR family members have been described and are
Fish & Richardson P.C.
Millennium Pharmaceuticals Inc.
Mosher Mary E.
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