Chitinase materials and methods

Details Chitinase materials and methods Chitinase materials and methods

1997-06-16

2002-04-16

Prouty, Rebecca E. (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S209000, C536S023200

Reexamination Certificate

active

06372212

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to human chitinase enzyme and more specifically to novel purified and isolated polynucleotides encoding human chitinase, to the chitinase products encoded by the polynucleotides, to materials and methods for the recombinant production of chitinase products and to antibody substances specific for the chitinase.
BACKGROUND
Chitin is a linear homopolymer of &bgr;-(1,4)-linked N-acetylglucosamine residues. This polysaccharide is second only to cellulose as the most abundant organic substance. The exoskeleton of arthropods is composed of chitin. In addition, fungi and other parasites contain chitin in their outer cell wall, where it serves important structural and protective roles. Disruption of the fungal cell wall and membrane has been a useful therapeutic strategy against fungi and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Despite the existence of anti-fungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See, Georgopapadakou et al.,
Trends Microbiol.,
3: 98-104 (1995). The fungal species and parasites responsible for these diseases are mainly Canrda, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
Chitin can be degraded by the enzyme chitinase. Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. Chitinases have been cloned from numerous bacterial [e.g.,
Serratia marcescens
, Jones et al.,
EMBO J.,
5:467-473 (1986)], plant [e.g., tobacco, Heitz et al.,
Mol. Gen. Genet.,
245:246-254 (1994)], and insect [e.g., wasp, Krishnan et al.,
J. Biol. Chem.,
269:20971-20976 (1994)] species.
Several proteins with low homology to bacterial, insect, and plant chitinases (less than 40% amino acid identity) have been identified in mammals, such as human cartilage gp-39 (C-gp39) [Hakala et al.,
J. Biol. Chem.,
268.25803-25810 (1993)], human glycoprotein YKL-40 [Johansen et al.,
Eur. J. Cancer,
31A: 1437-1442 (1995)], oviduct-specific, estrogen-induced protein from sheep [DeSouza et al.,
Endocrinology,
136:2485-2496 (1995)], cows and humans; and a secretory protein from activated mouse macrophages [Chang et al., Genbank M94584]. However, chitin-degrading activity has not been reported for these proteins. The function of these proteins is not known, but they have been postulated to be involved in tissue remodeling. Hakala et al., supra, report that C-gp39 is detectable in synovial and cartilage specimens from rheumatoid arthritis patients, but not from normal humans. Recklies et al.,
Athritis Rheumatism,
36(9 SUPPL.):S190 (1993) report localization of the C-gp39 protein to a distinct population of cells in the superficial layers of cartilage. Johansen et al., supra, report that measurements of YKL-40 serum levels are of value as a potential prognostic marker for the extent of metastatic disease and survival of patients with recurrent breast cancer.
Escott et al.,
Infect. Immun.,
63:4770-4773 (1995) demonstrated chitinase enzymatic activity in human leukocytes and in human serum. Overdijk et al.,
Glycobiology,
4:797-803 (1994) described isolation of a chitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase) from human serum and rat liver. Renkema et al.,
J. Biol. Chem.,
270:2198-2202 (February 1995) prepared a human chitotriosidase from the spleen of a Gaucher disease patient. Their preparation exhibited chitinase activity and the article reports a small amount of amino acid sequence of the protein component of the preparation (22 amino terminal residues and 21 residues of a tryptic fragment). The function of human chitinase is also unknown, but a relationship with the pathophysiology of Gaucher disease is proposed in the article. A later publication by the same group [Boot et al.,
J. Biol. Chem.,
270(44):26252-26256 (November 1995)] describes the cloning of a human macrophage cDNA encoding a product that exhibits chitinase activity. The partial amino acid sequence reported by the group in their February 1995 article matches portions of the deduced amino acid sequence of the human macrophage cDNA product. See also International Patent Publication No. WO 96/40940.
In view of the increasing incidence of life-threatening fungal infection in immunocompromised individuals, there exists a need in the art to identify and isolate polynucleotide sequences encoding human chitinases, to develop materials and methods useful for the recombinant production of the enzyme, and to generate reagents for the detection of the chitinase in plasma.
SUMMARY OF THE INVENTION
The present invention provides novel purified and isolated polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding human chitinase or fragments and analogs thereof; methods for the recombinant production of chitinase polypeptides, fragments and analogs thereof; purified and isolated chitinase polypeptide fragments and analogs; antibodies to such polypeptides, fragments and analogs; and pharmaceutical compositions comprising these polypeptides, fragments, analogs, or antibodies.
Specifically provided are: purified, isolated polynucleotides encoding the human chitinase amino acid sequence of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof; DNAs comprising the protein coding nucleotides of SEQ ID NOS: 1 or 3, particularly nucleotides 65 to 1402 of SEQ ID NO: 1 or nucleotides 90 to 1427 of SEQ ID NO: 3; purified, isolated polynucleotides comprising a polynucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 14 or 15; purified, isolated polynucleotides encoding human chitinase selected from the group consisting of: (a) a double-stranded DNA comprising the protein coding portions of the sequence set out in either SEQ ID NO: 1 or SEQ ID NO: 3, (b) a DNA which hybridizes under stringent conditions to a non-coding strand of the DNA of (a), and (c) a DNA which, but for the redundancy of the genetic code, would hybridize under stringent conditions to a non-coding strand of DNA sequence of (a) or (b); vectors comprising such DNAs, particularly expression vectors wherein the DNA is operatively linked to an expression control DNA sequence; host cells stably transformed or transfected with such DNAs in a manner allowing the expression in said host cell of human chitinase; a method for producing human chitinase comprising culturing such host cells in a nutrient medium and isolating human chitinase from said host cell or said nutrient medium; purified, isolated polypeptides produced by this method; purified, isolated polypeptides comprising the human chitinase amino acid sequence of SEQ ID NOS: 2 or 4, particularly amino acids 1 to 445 thereof; human chitinase fragments lacking from 1 to about 72 C-terminal amino acid residues of mature human chitinase, particularly the human chitinase fragment of SEQ ID NO: 14; the human chitinase analog of SEQ ID NO: 15; hybridoma cell lines producing a monoclonal antibody that is specifically reactive with one of the above-described polypeptides; and monoclonal antibodies produced by such hybridomas.
Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The nucleotide sequence of two human cDNAs encoding presumed allelic variants of human chitinase, and including noncoding 5′ and 3′ sequences, are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. These DNA sequences and DNA sequences which hybridize to the noncoding strand t

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