Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1998-01-07
2000-04-25
Hobbs, Lisa J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
435366, 4352523, 43525411, 530412, C12N 910, C12N 500, C12N 120, A23J 100
Patent
active
060543043
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to an .alpha.1-6 fucosyltransferase derived from pig or human. More particularly, the present invention relates to a novel .alpha.1-6 fucosyltransferase derived from human, which is an enzyme that transfers fucose from guanosine diphosphate (GDP)-fucose by .alpha.1.fwdarw.6 linkage to N-acetylglucosamine (GlcNAc) bound to Asn at the stem of asparagine type sugar chain (Asn type sugar chain) and which is useful in the field of glyco-technology for modification and synthesis of sugar chain and/or for the diagnosis of diseases such as malignant tumor, and to a gene encoding said enzyme.
BACKGROUND ART
The structure and function of sugar chain moiety of complex carbohydrates, such as glycoprotein and glycolipid, derived from higher organisms have been drawing much attention in recent years, and many studies are under way. While a sugar chain is formed by the action of glycohydrolase and glycosyltransferase, glycosyltransferase contributes greatly to its formation.
Using a sugar nucleotide as a sugar donor, glycosyltransferase transfers a sugar to a receptor sugar chain, thereby to elongate the sugar chain. The specificity for the structure of receptor sugar chain is stringent, such that one glycoside linkage is formed by the corresponding one transferase. Hence, glycosyltransferases are used for structural studies of sugar moiety of complex carbohydrate, for facilitated synthesis of a particular sugar chain structure, and for modification of native sugar chain structure.
Besides, glycosyltransferases are expected to be usable for the modification of the nature of complex carbohydrate and cells, by means of artificial alteration of sugar chain. For this end, the development of various glycosyltransferases having identified substrate specificity has been awaited.
An .alpha.1-6 fucosyltransferase is an important enzyme found in Golgi appratus of organelle, which is considered to be one of the enzymes that control processing of asparagine-linked sugar chain. Therefore, the enzyme will be useful for the elucidation of control mechanism and control of formation of sugar chain structure, once acted on an asparagine-linked sugar chain.
In addition, the activity of .alpha.1-6 fucosyltransferase and the proportion of reaction products of this enzyme are known to increase in certain diseases such as liver cancer and cystic fibrosis. Therefore, a rapid development of the method for diagnosis of these diseases has been desired, which involves determination of the activity of this enzyme, Northern blot using a cDNA encoding .alpha.1-6 fucosyltransferase, or RT-PCR assay of mRNA amount transcribed and expressed in the living body.
The activity of .alpha.1-6 fucosyltransferases has been detected in body fluids or organs of various animals and culture cells thereof, and there has been known, as a purified enzyme product, an enzyme derived from human cystic fibrosis cell homogenates [Journal of Biological Chemistry, vol. 266, pp. 21572-21577 (1991)]. According to this report, however, the enzyme is associated with drawbacks in that (1) its optimum pH is 5.6 which is different from physiological pH, (2) it has relatively low molecular weights (34,000 and 39,000) by SDS-polyacrylamide gel electrophoresis, (3) its large scale and stable supply is practically unattainable due to its being derived from human cell, and others.
This enzyme is obtained as a membrane-bound enzyme, and requires bovine serum for culturing the cells, which in turn results in difficult purification of the enzyme and a huge amount of money necessary for culture of the cells to be a starting material. Consequently, stable supply of this enzyme preparation is all but impractical.
While a chemical synthesis is often employed for synthesizing a sugar chain, the synthesis of oligosaccharides requires many steps that have been necessitated by its complicated synthesis route and specificity of the reaction, so that it involves various practical problems. Particularly, binding of fucose to GlcNAc bound to Asn of aspa
REFERENCES:
1. J. Biol. Chem. 271(44) 1996 Uozimu N. et al.: "Purification and cDNA cloning of porcine brain" pp. 27810-27817.
2. J. Biochem. 120(2) 1996 Uozume N. et al. "A fluorescent assay method for GDP-L-Fuc:N-acetyl- beta-D-glucosaminide alpha-1-6 fucosyltransferase activity involving high performance liquid chromatography", pp. 385-392.
3. J. Biol. Chem. 266(32) 1991 Voynow J. A. et al., "Purification and characterization of GDP-1-fucose-N-acetyl-beta-D-glucosaminide alpha-1-6 fucosyltransferase from cultured human skin fibroblasts requirement of a specific biantennary oligosaccharide as substrate", pp. 21472-21577.
Shiba Tetsuo
Taniguchi Naoyuki
Uozumi Naofumi
Yanagidani Shusaku
Hobbs Lisa J.
Toyo Boseki Kabushiki Kaisha
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