Chemistry: molecular biology and microbiology – Vector – per se
Patent
1993-04-30
2000-10-03
Teng, Sally P.
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 435243, 435325, 514 2, 530350, 536 235, C12N 100, C12N 1563
Patent
active
061271691
DESCRIPTION:
BRIEF SUMMARY
The invention relates to proteins known as .beta.-galactoside binding proteins and to their use to arrest, control or otherwise affect cell growth.
Proteins capable of binding specific sugars on cell surfaces have been known for many years. The sugar binding proteins hitherto known are more commonly referred to by the generic term "lectin". A lectin is defined as a carbohydrate binding protein which has a specificity for particular sugar residues, is bivalent or polyvalent with respect to sugar binding and is at least bivalent if monomeric (Barondes, H, Science 223, 1259-1264 (1984)). Due to the multivalency it is a further characterising property of lectins that they cause agglutination of red blood cells i.e. they are haemagglutinins and can also cause agglutination of other cells because of their affinity for sugars on the cell surface. Also, because of their cross-linking property, which relates to bivalency or polyvalency they can impede cell proliferation. However their effect is indiscriminate, physiologically irreversible and they are highly toxic.
Particularly well-known examples of lectins are the plant proteins Concanavalin A and phytohaemagglutinin. More recently, sugar binding proteins have been identified in many other organisms, including vertebrates and slime moulds. Vertebrate proteins in this class have been described and characterised, for example, in the following references: and Kasai, K. Biochem. Biophys. Res. Comm. 134, 51-56 (1986) Lett. 214, 301-304 (1987) Massaro, D. Biochemistry 27, 692-699 (1988) McGrogan, M. and Nedwin, G. E. J. Biol. Chem. 264, 1310-1316 (1989)
It will be apparant from the above definition of a lectin that, as .beta.-galactosides are sugars, .beta.-galactoside binding proteins fulfil one of the criteria of the lectins and it was hitherto understood that all .beta.-galactoside binding proteins were in fact lectins. It is demonstrated herein by the present inventors that this is in fact not the case.
Plant lectins have been known to cause lymphocyte activation. EP-A-0337299 describes also the use of lectins from a variety of vertebrate sources in the treatment of diseases involving defects in the immune system such as myasthenia gravis and reumatoid arthritis. A beneficial effect on diabetes and multiple sclerosis is also suggested.
In EP-A-0260497 a "lectin-like" protein isolated from a cell line of Sarcophaga peregrina (flesh fly) is described which acts as a haemagglutinin and which has an inhibitory effect on the growth of tumours in mice.
The above documents' claims are however based on the lectin characteristics of the proteins used. The present inventors, in the search for new and improved cytostatic agents, have now isolated, purified and characterised a new cytostatic protein capable of inhibiting or arresting the growth of both normal and cancer cells, which is produced by mouse embryo fibroblasts and which does not fulfil the criteria for a lectin nature. By determination of its amino acid sequence and comparison of the sequence of isolated peptides with the known sequences of other proteins using literature and database searches it has been established that the inhibitor is a .beta.-galactoside binding protein (GBP). This has been confirmed by cDNA cloning and expression of the protein in recombinant form.
It has further been discovered by the inventors that the protein is not a lectin in accordance within the classical definition because it is monomeric and because it is monovalent with respect to sugar binding sites or it has no available sugar binding site because it is masked by a glycan complex. It is thus incapable of causing blood cells or other cells to agglutinate. Furthermore it does not exist in dimeric form as reported for the lectins but rather as a native monovalent monomer. Alternatively, it can form tetramers in which the four .beta.-galactoside binding sites are internal and thus not available to cause cell agglutination, even where no glycan complex is associated with the molecule. Furthermore the cytostatic effect is not exerted th
REFERENCES:
Kajikawa, T., et al., "Release of Cytotoxin By Macrophages on Treatment with Human Placenta Lectin", Life Sciences, 39:1177-1181 (1986).
Hynes, M. A., et al., "Selective Expression of an Endogenous Lactose-Binding Lectin Gene in Subsets of Central and Peripheral Neurons", The Journal of Neuroscience, 10(3) :1004-1013 (1990).
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Clerch, L. B., et al., "Sequence of a Full-Length cDNA for Rat Lung .beta.-Galactoside-Binding Protein: Primary and Secondary Structure of the Lectin", Biochemistry, 27:692-699 (1988).
Couraud, P-O., et al., "Molecular Cloning, Characterization, and Expression of a Human 14-kDa Lectin", The Journal of Biological Chemistry, 264(2) :1310-1316 (1989).
Greene, W. C., et al., "Soluble Suppressor Supernatants Elaborated by Concanavalin A-Activated Human Mononuclear Cells", The Journal of Immunology, 126(3) : 1185-1191 (1981).
Chany-Fournier, F., et al., "Action inhibitrice sur le developpement de la tumeur 180/TG de Crocker de l'antagoniste tissuelaire de l'interferon : lectine murine nouvellement reconnue", C. R. Acad. Sc. Paris, t.286:1550-1553 (1978).
Drickamer, K., et al., "Two Distinct Classes of Carbohydrate-recognition Domains in Animal Lectins", The Journal of Biological Chemistry, 263(20) :9557-9560 (1958).
Hirabayashi, J., et al., "Complete Amino Acid Sequence of a .beta.-Galactoside-Binding Lectin from Human Placenta", J. Biochem., 104:1-4 (1988).
Horabayashi, J., et al., "Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa .beta.-galactoside-binding lectin", Biochimica et Biophysica Acta, 1008:85-91 (1989).
Wells et al., Journal of Cellular Physiology, vol. 117, p. 148, 1983 .
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Barondes, Science, 233, 1259-1264, 1984.
Mallucci Livio
Wells Valerie
Brezner David J.
Teng Sally P.
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