Dendritic cell transmembrane serine protease

Details Dendritic cell transmembrane serine protease Dendritic cell transmembrane serine protease

2002-06-20

2004-09-21

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C530S324000

Reexamination Certificate

active

06794173

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to Dendritic Cell Transmembrane Serine Protease (DCTSP), a new member of the Type II Transmembrane Serine Protease polypeptide family, and to methods of making and using DCTSP polypeptides.
BACKGROUND OF THE INVENTION
Type II Transmembrane Serine Protease (TTSP) polypeptides are related, membrane-anchored polypeptides that are involved in cell surface proteolysis and share common structural features including a proteolytic domain, a stem region comprising varying modular structural domains, a transmembrane domain, and a short cytoplasmic domain (Hooper et al.,
J. Biol. Chem
. 276:857, 2001). Members of this family include hepsin (Leytus et al.,
Biochemistry
27:1067, 1988), enteropeptidase (also referred to as enterokinase; Kitamoto et al.,
Biochemistry
34:4562, 1995), TMPRSS2 (Paoloni-Giacobino et al.,
Genomics
44:309 1997), human airway trypsin-like protease (HAT; Yamaoka et al.,
J. Biol Chem
. 273:11895, 1998), corin (Yan et al.
J. Biol. Chem
. 274:14296, 1999), MT-SP1 (also known as matriptase; Lin et al.,
J. Biol. Chem
. 274:18231, 1999), and TMPRSS4 (Wallrapp et al.,
Cancer Res
. 60:2602, 2000). Kim et al. (
Biochim. Biophys. Acta
. 1518:204, 2001) disclose cDNAs encoding proteins with putative serine protease domains and potential regulatory domains; one of the putative proteins also had a transmembrane domain.
The proteolytic domains of TTSPs exhibit a high degree of homology, with highly conserved motifs comprising histidine, aspartate and serine residues thought to be necessary for catalytic activity. A conserved activation motif contains an arginine or lysine, and indicates that the TTSPs are likely to be activated following cleavage. The presence of conserved cysteine residues, and their predicted disulphide bonding pattern, provide support for the belief that TTSPs are likely to remain associated with the cell membrane even after cleavage/activation, although soluble forms of some TTSPs have been identified (Hooper et al., supra). Additional conserved cysteine residues appear to be involved in forming disulphide bonds within the catalytic domain. Cleavage specificities and potential substrates have been identified for some TTSPs, but remain unknown for most; it is likely that the substrate(s) for TTSPs preferentially contain an arginine or lysine in the P1 amino acid position (as originally described for serine proteases in Schecter et al.,
Biochem. Biophys. Res. Commun
. 27:157, 1967).
A hydrophobic, transmembrane domain is present near the N-terminus of the members of the TTSP family, indicating that the proteolytic domain is extracellular. The presence of the catalytic domain on the outside of cell, but presumably still in association with the cell membrane, suggests a role for this family of serine proteases in regulated release of substrate proteins from the cell surface, either from the same cell upon which the TTSP is found or from a cell that is in close association with such a cell.
Most TTSPs exhibit relatively restricted expression patterns, indicating that they may carry out tissue-specific functions (Hooper et al., supra). The variability of the length of the cytoplasmic domains of TTSPs renders it difficult to predict a role for these proteins in cellular signaling; however, some TTSPs do contain consensus phosphorylation sites within the cytoplasmic domain. The greatest degree of variability between members of the TTSP family occurs in the stem region, which may contain up to eleven structural domains. The variety in number and type of structural domains present in the stem region suggests that it may serve to regulate the activity and/or binding of TTSPs to substrates. The role of cell surface proteolysis in homeostasis and disease demonstrates that there is a need in the art to identify and characterize additional members of the TTSP family.
SUMMARY OF THE INVENTION
The present invention provides a novel serine protease, referred to as human Dendritic Cell Transmembrane Serine Protease (DCTSP), a Type II transmembrane protein depicted in SEQ ID NOs:1 and 2; a splice variant having 446 amino acids was also isolated; the nucleotide and amino acid sequence of this variant are shown in SEQ ID NOs:3 and 4. Also provided are DCTSP polypeptides that are encoded by nucleic acids capable of hybridizing to the DNAs of SEQ ID NOs:1 or 3 and that have at least one DCTSP activity, DCTSP polypeptides that are at least 80% identical to the DCTSP polypeptides of SEQ ID NOs: 2 or 4 and that have at least one DCTSP activity, and fragments of such DCTSP polypeptides that have at least one DCTSP activity.
The invention further provides various forms of DCTSP, including: a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 207 to 212, inclusive, and y represents an integer from 470 to 477, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 221 to 226, inclusive; a protein comprising amino acids x to y of SEQ ID NO:2, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 75 to 95, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 1 to 47, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 174 to 179, inclusive, and y represents an integer from 441 to 446, inclusive; a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 174 to 179, inclusive, and y represents an integer from 190 to 195, inclusive; and a protein comprising amino acids x to y of SEQ ID NO:4, wherein x represents an integer from 91 to 96, inclusive, and y represents an integer from 190 to 195, inclusive. Also comprehended herein are nucleic acids encoding the aforementioned DCTSP polypeptides, vectors comprising such nucleic acids, host cells transformed or transfected with such vectors (including hosts cells wherein the DNA encoding a DCTSP polypeptide is integrated into host cell chromosomal DNA), and processes for obtaining DCTSP polypeptides by culturing such host cells.
Soluble forms of DCTSP (including fragments comprising the extracellular domain as well as fragments comprising the cytoplasmic domain) will be useful in vitro to screen for agonists or antagonists of DCTSP activity utilizing one or more screening methods, which methods also form an aspect of the present invention. In one aspect, the inventive methods utilize homogeneous assay formats such as fluorescence resonance energy transfer, fluorescence polarization, time-resolved fluorescence resonance energy transfer, scintillation proximity assays, reporter gene assays, fluorescence quenched enzyme substrate, chromogenic enzyme substrate and electrochemiluminescence. In another aspect, the inventive methods utilize heterogeneous assay formats such as enzyme-linked immunosorbant assays (ELISA) or radioimmunoassays. In yet another aspect of the invention are cell-based assays, for example those utilizing reporter genes, as well as functional assays that analyze the effect of an antagonist or agonist on biological function(s).
The invention further provides methods for producing information comprising the identity of a compound that alters one or more activities of DCTSP, comprising using one or more of the inventive assays to identify a compound or compounds that alter the binding and/or cleavage of substrate

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