Polynucleotides encoding a novel metalloprotease, MP-1

Details Polynucleotides encoding a novel metalloprotease, MP-1 Polynucleotides encoding a novel metalloprotease, MP-1

2002-02-05

2003-11-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S252300, C435S320100, C435S219000, C536S023200

Reexamination Certificate

active

06642041

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides novel polynucleotides encoding MP-1 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel MP-1 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.
BACKGROUND OF THE INVENTION
Proteases hydrolyze specific peptide bonds in proteins. The residues at the active site are used to classify proteases (Rawlings & Barrett, 1995). Proteases that hydrolyze peptide bonds using metal ions are referred to as metalloproteases (“MP”). The metalloproteinases may be one of the older classes of proteases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures, but many contain a zinc ion. In some cases, zinc may be replaced by another metal such as cobalt or nickel.
The gene of the present invention encodes a human protease belonging to the peptidase m22 family (see Rawlings & Barrett, 1995 for review of protease familial classification). This family contains the sequence HHMEAH (SEQ ID NO:24) The histidine and/or glutamic acid within this sequence are thought to coordinate metal ion binding. Such metal ion coordination facilitates catalysis through the stabilization of a noncovalent, tetrahedral intermediate after the attack of a metal-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group. Metal ion coordination is thought to stabilize the negative charges formed within the active site of the enzyme during catalysis. Such stabilization lowers the transition state energy requirements, and thus results in significant rate enhancements during enzymatic catalysis over non-metal ion coordination conditions (Fersht, A., “Enzyme Structure and Mechanism”, 2
nd
edition, W. H. Freeman and Company, New York, 1985).
The prototype of this family is a secreted O-sialoglycoprotein endopeptidase (so called because it has specificity for cleavage of proteins which contain O-sialoglycans attached to serine and threonine residues) from the bacterium
pasteurella haemolytica
(Abdullah et al., 1991; Mellors and Lo, 1995). Substrate proteins for the
P. haemolytica
O-sialoglycoprotein endopeptidase include the cell plasma membrane glycoproteins glycophorin A and leukocyte surface antigens CD34 (expressed on stem cells in the bone marrow), CD43 (a sialomucin implicated in immune cell function and cell signaling), CD44 (a cell receptor for hyalurnate of extracellular matrix) and CD45 (involved in leukocyte activation) (Mellors & Lo, 1995). Other receptors cleaved by this protease include the counter receptor for P-selectin, the counter receptor for L-secton, the receptor for interleukin 7 and epitectin (Mellors & Lo, 1995).
Although the
P. haemolytica
O-Sialoglycoprotein endopeptidase is the best characterized protease of the peptidase m22 class, genes encoding for other family members have been identified in the genomic sequences of
Saccharomyces cerevisiae
(baker's yeast),
Borrelia burgdorferi
(lyme disease spirochete),
Escherichia coli, Helicobacter pylori,
Mycobacterium,
Haemophilus influenzae
and the cyanobacterium Synechocystis. In addition similar sequences have been identified from mycoplasma genitalium, mycoplasma pneumoniae, archaeoglobus fulgidus, pyrococcus horikoshii, chlamydia trachomatis, streptomyces coelicolor,
mycobacterium tuberculosis, mycobacterium leprae
and
bacillus subtilis.
The MP-1 gene of the present invention represents the first described mammalian homologue belonging to the m22 class of metalloproteinases.
Metalloproteinases in Disease
Limited-proteolysis by metalloproteases plays a central regulatory role in many physiological and pathophysiological processes. There are many examples of inhibitors of metalloproteases that are useful medications in the treatment of hypertension, heart failure, various forms of cancer and other diseases.
Metalloproteases play many important biological roles in the nervous system, including the spinal cord. There is a balance between the synthesis and degradation of extracellular matrix proteins in the process of synapse formation during development and regeneration. The timing of MP activation is therefore potentially critical. Some MPs have been shown to be upregulated in the spinal cord either during development or in pathological states such as multiple sclerosis, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis. Since MPs degrade extracellular matrix proteins, they would be toxic to developing neurons that depend upon the matrix proteins for survival, neurite outgrowth, and synapse formation. Degradation of the matrix proteins would also cause the breakdown of the blood brain barrier and infiltration of immune cells into the CNS, which occurs in inflammatory conditions such as MS.
Using the above examples, it is clear the availability of a novel cloned metalloproteinase provides opportunities for adjunct or replacement therapy, and are useful for the identification of metalloproteinase agonists, or stimulators (which might stimulate and/or bias metalloproteinase action), as well as, in the identification of metalloproteinase inhibitors. All of which might be therapeutically useful under different circumstances. The metalloproteinase of the present invention can also be used as a scaffold to tailor-make specific metalloproteinase inhibitors.
Polynucleotides and polypeptides corresponding to a portion of the full-length MP-1 polypeptide of the present invention, in addition to its encoding polynucleotides, have been described by Chen et. al. and is described as a putative sialoglycoprotease type 2 protein (Genbank Accession No. gi|11641265).
The protein referenced in Genbank Accession No. gi|11641265 appears to represent an aberrant splice variant of the MP-1 polypeptide of the present invention, having an additional 25 amino acids inserted after position 273 of MP-1 (SEQ ID NO:2). In addition, there are several significant amino acid differences. At amino acid position 31 of SEQ ID NO:2, MP-1 contains a Glycine amino acid (“G”) instead of the Glutamic amino acid (“E”) referenced in Genbank Accession No. gi|11641265. And at amino acid position 373 of SEQ ID NO:2, MP-1 contains an Alanine amino acid (“A”), as opposed to the Glycine amino acid (“G”) referenced in Genbank Accession No. gi|11641265.
Confirmation that the MP-1 polypeptide sequence of the present invention is the correct sequence, and that the additional 25 amino acids observed within the gi|11641265 protein sequence is a result of aberrant splicing became apparent through the application of several bioinformatics methods. First, an analysis was performed to evaluate the polynucleotide and polypeptide sequence of MP-1 of the present invention to its corresponding genomic sequence (Genbank Accession No. gi|AC013468) as shown in FIG.
6
. When AC013468 was analyzed using the GenewiseDB program against MP-1 of the present invention, both the G31 and E373 amino acids were present in the genomic sequence (as shown in FIG.
6
). Furthermore, a splice exon/intron splicing junction was clearly present near position 273 of MP-1. In addition, the 25 amino acid insertion observed in gi|11641265 was considered an intron by the GenewiseDB program. When gi|11641265 was analyzed against AC013468 using GenewiseDB, the presence of the 25 amino acid insertion caused a frameshift in the coding sequence. Thus, the 25 amino acid insertion present in gi|11641265 represents an unspliced intron in the cDNA sequence. Thus, based upon the indic

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