Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-15
2004-07-06
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S252330, C435S320100, C435S810000, C536S023500
Reexamination Certificate
active
06759194
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to a new peroxisome-associated polypeptide, the nucleotide sequence encoding said polypeptide and portions thereof as well as their uses in the diagnosis of several diseases, especially the diagnosis and/or the treatment of lung injuries and diseases, and of oxidative stress-related disorders.
BACKGROUND OF THE INVENTION
The peroxisomes are organelles nearly ubiquitous in eukaryotic cells. They contain enzymes essential for various catabolic and anabolic pathways. Some of these enzymes are expressed constitutively while others can be induced under appropriate conditions. Peroxisomes carry out a variety of essential reactions such as peroxisomal oxidation and respiration, fatty acid beta-oxidation, cholesterol and dolichol metabolism, ether-phospholipid synthesis, and glyoxylate and pipecolic acid metabolism.
The peroxisomal respiratory pathway is based upon the formation of hydrogen peroxide by a collection of oxidases and the decomposition of the H
2
O
2
by catalase These reactions are responsible for 20% of oxygen consumption in liver, and several oxidases have been identified in peroxisomes. Ethanol elimination via catalase in peroxisomes may be significant in addition to the oxidation via cytosolic alcohol dehydrogenase.
The peroxisomal beta-oxidation system catalyses the beta-oxidative chain shortening of a specific set of compounds which can not be handled by mitochondria: very long chain fatty acids, di- and trihydroxycholestanoic acids, pristanic acid, long chain dicarboxylic acids, several prostaglandins, several leukotrienes, 12- and 15-hydroxyeicosatetraeonic acid, and several mono- and polyunsaturated fatty acids, which are of direct diagnostic relevance for some peroxisomal disorders.
Peroxisomes play also a major role in the synthesis of cholesterol and other isoprenoids. Fibroblasts from patients affected by disorders of peroxisome biogenesis show low capacity to synthesise cholesterol.
Two enzyme activities responsible for introduction of the characteristic ether linkage in ether-linked phospholipids (dihydroacetonephosphate acyltransferase (DHAPAT) and alkyldihydroxyacetonephosphate synthase (alkyl-DHAP synthase)) are localised in peroxisomes. These enzymes are not yet cloned. As demonstrated by the identification of patients with deficiency of either DHAPAT or alkyl-DHAP synthase with severe clinical abnormalities, ether-phospholipids are of major importance in humans.
Peroxisomes are able to detoxify glyoxylate via alanine/glyoxylate aminotransferase. The deficiency of this cloned enzyme causes hyperoxaluria type I. L-pipecolate is a minor metabolite of L-lysine and is catabolised by the L-pipecolate oxidase localised in peroxisomes. The enzyme is deficient in cerebro-hepato-renal (Zellweger) syndrome.
In human, the importance of peroxisomes was emphasised by a number of inherited diseases involving either a defect in the biogenesis of peroxisomes or a deficiency of one (or more) peroxisomal enzymes. So far, 12 different peroxisomal disorders have been described and most of them are lethal.
A wide variety of chemicals have been showed to produce peroxisome proliferation and induction of peroxisomal and microsomal fatty acids-oxidising enzymes activities in rats and mice. Several peroxisomes proliferators have been shown to increase the incidence of liver tumours in these species. Proposed mechanisms of liver tumour formation by peroxisomes proliferators include induction of sustained oxidative stress.
Therefore, newly identified molecules associated with peroxisomes could be used for the development of diagnostic tools and possibly for the improvement of several therapeutical applications of various diseases associated with peroxisomal disorders. In addition, it is useful to identify the molecules present in specific organs like the lung and which may be used as specific markers of inflammatory diseases as well as lung injuries or diseases.
SUMMARY OF THE INVENTION
The Inventors have isolated and purified a new sequence of a low molecular weight human broncho-alveolar polypeptide. Said mammal, preferably human, protein or polypeptide (hereafter identified as B18hum protein) has been sequenced and its corresponding genomic DNA (SEQ ID NO 8) and cDNA (SEQ ID NO 1) have been identified. Similarly, the corresponding nucleotide and amino acid sequence from a rat (SEQ ID NO 3 and 4) and from a mouse (SEQ ID NO 5 and 6) have been obtained.
Said sequences present several homologies with other peroxisomal proteins of yeast and comprise a carboxy-terminal tripeptide SQL which is necessary for the specific targeting and translocation of several proteins into the peroxisome.
Therefore, the present invention is related to a new isolated and purified polypeptide sequence having a amino acid sequence which presents more than 70% homology, advantageously more than 85% homology, more preferably more than 95% homology, with the amino acid sequence SEQ ID NO 2. Said amino acid sequence is advantageously obtained from a mammal, preferably from a rat, a mouse or a human.
The present invention is also related to the isolated and purified polypeptide sequence corresponding to the amino acid sequence SEQ ID NO 2 or a portion thereof, preferably an immunoreactive portion (putative immunogenic domain or T or B cell epitopes).
Said portions are advantageously comprised between:
Glutamic acid position 14—Glutamic acid position 28
Alanine position 27—Leucine position 37
Alanine position 43—Glutamic acid position 58
Glutamic acid position 58—Valine position 70
Valine position 81—Leucine position 98
Arginine position 96—Leucine position 113
Serine position 119—Serine position 130
Valine position 138—Threonine position 151
Preferably, said portion has more than 10, 20, 30, 50 or 70 amino acids. Specific portions of the amino acid sequence SEQ ID NO 2 are also portions of more than 70 amino acids which present at least 80% of the proteinic activity (see example 5) of the complete SEQ ID NO 2 sequence. Therefore, the amino acid sequence according to the invention can be partially deleted while maintaining its activity, preferably its anti-oxidative activity, which will be described hereafter.
According to the invention, the amino acid sequence SEQ ID NO 2 presents a pI of 7.16 and a molecular weight of 17047 Dalton as hereafter defined by bidimensional electrophoresis.
The present invention is also related to the nucleotide sequence encoding the amino acid sequence according to the invention and its regulatory sequences upstream said coding sequence. A nucleotide sequence encoding the polypeptide according to the invention is a genomic DNA (see SEQ ID NO 10), a cDNA (see SEQ ID NO 1) or a mRNA, possibly comprising said upstream regulatory sequence. Advantageously, said nucleotide sequence presents more than 70%, advantageously more than 85%, more preferably more than 95% homology with SEQ ID NO 1 or its complementary strand.
According to a preferred embodiment of the present invention, said nucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 1, its complementary strand or a portion thereof.
“A portion of the nucleotide sequence SEQ ID NO 1” means any nucleotide sequence of more than 15 base pairs (such as a primer, a probe or an antisense nucleotide sequence) which allow the specific identification, reconstitution or blocking of the complete nucleotide sequence SEQ ID NO 1 (including its regulatory sequences upstream the coding sequence).
Said portions allow the specific identification, reconstitution or blocking by specific hybridisation with the nucleotidic sequence SEQ ID NO 1, preferably under standard stringent conditions, with sequences like probes or primers possibly labelled with a compound (radioactive compound, enzyme, fluorescent marker, etc.), and can be used in a specific diagnostic or dosage method like probe hybridisation (see Sambrook et al., §§9.47-9.51 in
Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y. (1989)), genetic amplification (like PCR (
Bernard Alfred
Falmagne Paul
Hermans Cedric
Knoops Bernard
Wattiez Ruddy
Chan Christina
Huynh Phuong
Knobbe Martens Olson & Bear LLP
Universite Catholique de Louvain
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