&agr;1-6 fucosyltransferase

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease

Reexamination Certificate

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C435S183000, C435S069100, C435S252300, C435S320100, C536S023100, C536S023200

Reexamination Certificate

active

06291219

ABSTRACT:

TECHNICAL FIELD
The present invention relates to an &agr;1-6 fucosyltransferase derived from pig or human. More particularly, the present invention relates to a novel &agr;1-6 fucosyltransferase derived from human, which is an enzyme that transfers fucose from guanosine diphosphate (GDP)-fucose by &agr;1→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 &agr;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 &agr;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 &agr;1-6 fucosyltransferase, or RT-PCR assay of mRNA amount transcribed and expressed in the living body.
The activity of &agr;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 asparagine-linked sugar chain by &agr;1→6 linkage is extremely difficult due to the instability of fucose.
DISCLOSURE OF THE INVENTION
It is therefore an object of the present invention to stably provide an &agr;1-6 fucosyltransferase in large amounts, which is useful as a reagent for structural analysis of sugar chain or glyco-technology, or as diagnostics.
Another object of the present invention is to provide a method of producing &agr;1-6 fucosyltransferase in large amounts by the use of a human- or porcine-derived &agr;1-6 fucosyltransferase gene. It is aimed to use such specific genes so as to enable development of a method for diagnosis of diseases by Northern blot using a DNA encoding said enzyme, or by RT-PCR assay of mRNA amount transcribed and expressed in the living body.
In an attempt to achieve the above-mentioned objects, the present inventors started the study of an enzyme capable of linking fucose to GlcNAc linked to Asn of asparagine type sugar chain by &agr;1→6 linkage, using a fluorescence-labeled substrate analogous to an asparagine type sugar chain which is a receptor of this enzyme. As a result, they have found the activity of this enzyme in the extract fractions of porcine brain which is readily available as a starting material to be purified, and they have purified said enzyme from said fractions and elucidated the enzymatic and physico-chemical properties, which resulted in the completion of the invention.
Accordingly, the present invention relates to a porcine-derived &agr;1-6 fucosyltransferase having the following physico-chemical properties (hereinafter this enzyme is referred to as porcine &agr;1-6 fucosyltransferase).
(1) Action: transferring fucose from guanosine diphosphate-fucose to the hydroxy group at 6-position of GluNAc closest to R of a receptor (GlcNAc&bgr;1-2Man&agr;1-6) (GlcNAc&bgr;1-2Man&agr;1-3)Man&bgr;1-4GlcNAc&bgr;1-4GlucNAc-R wherein R is an asparagine residue or a peptide chain carrying said residue, whereby to form (GlcNAc&bgr;1-2Man&agr;1-6)- (GlcNAc&bgr;1-2Man&agr;1-3)Man&bgr;1-4GlcNAc&bgr;1-4(Fuc&agr;1-6)GlucNAc-R.
In the above formula, asparagine residue at R is a residue wherein the acid amide group at the side chain of asparagine is bound to the hydroxy group at the anomer position of the reducing terminal of sugar chain, and a peptide chain having said residue is a peptide chain having said residue in the peptide to which two or more amino acids are bound, which is preferably a peptide chain having —Asn—(X)—Ser/Thr—.
(2) optimum pH: about 7.0
(3) pH stability: stable in the pH range of 4.0-10.0 by treatment at 4° C. for 5 hours
(4) optimum temperature: about 30-37° C.
(5) inhibition or activation: no requirement for divalent metal ion for expression of activity; no inhibition of activity even in the presence of 5 mM EDTA
(6) molecular weight: about 60,000 by SDS-polyacrylamide gel electrophoresis.
The present inventors have purified &agr;1-6 fucosyltransferase alone from porcine brain, analyzed the amino acid sequence of this protein and cloned a gene based on the partial amino acid sequence to accomplish the present invention.
That is, the present invention provides a gene encoding porcine &agr;1-6 fucosyltransferase.
The present invention also provides an expression vector containing a gene encoding porcine &agr;1-6 fucosyltransferase.
The present invention further provides a transformant cell obtained by transforming a host cell with an expression vector containing a gene encoding porcine &agr;1-6 fucosyltransferase.
The present invention yet provides a method for producing a recombinant &agr;1-6 fucosyltransferase, comprising culturing a transformant cell obtained by transforming a host cell with an expression vector containing a gene encoding porcine &agr;1-6 fucosyltransferase, and harvesting the &agr;1-6 fucosyltransferase from the culture thereof.
The present inventors have reached the present invention by purifying protein having an &agr;1-6 fucosyltransferase activity from human cell culture broth and elucidating

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