Purified &bgr;1,2-xylosyltransferase and uses thereof

Details Purified &bgr;1,2-xylosyltransferase and uses thereof Purified &bgr;1,2-xylosyltransferase and uses thereof

2000-12-22

2003-07-15

Prouty, Rebecca E. (Department: 1652)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S069200, C435S183000, C435S193000, C536S022100, C536S023600, C536S024300, C536S024330

Reexamination Certificate

active

06593462

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of soybean enzymes and protein chemistry. More specifically, the present invention relates to purified &bgr;1,2-xylosyltransferase and uses thereof.
2. Description of the Related Art
Immediate hypersensitivity reactions to foods occurs in 6% to 8% of children, and about 1% of adults (1), and these reactions are mediated by the production of IgE antibodies to glycoproteins in these foods (2). The majority of allergies are to foods of plant origin, and a number of allergenic proteins have been identified from peanut, wheat, barley, rye and soy (3-7). While linear amino acid sequences and conformational structures of proteins have been identified as IgE-binding epitopes (8), there is increasing evidence (9-11) that specific carbohydrate structures may also be important as allergens.
While there is some similarity in peptide sequence between the different allergenic proteins, a common characteristic is that most contain N-linked oligosaccharides having a &bgr;1,2-linked xylose attached to the &bgr;-linked mannose of the core structure (12). Furthermore, in wheat and barley glycoproteins, xylose appears to be important for allergenicity, since those proteins containing a &bgr;1,2-xylose were more reactive in vivo and in vitro than deglycosylated proteins (13). In addition, IgE from allergic patients recognized an Endo-lys C peptide containing the glycan, and this recognition was lost upon deglycosylation (14).
The enzyme that adds xylose to these N-linked glycoproteins is the plant &bgr;1,2-xylosyltransferase. The identification of this enzyme in microsomes from
Phaseolus vulgaris
cotyledons (15), and from sycamore cells of
Acer pseudoplatanus
(16) has been reported, but there are no reports on the purification or properties of the enzyme. The substrate specificity for acceptor oligosaccharide was examined with particulate enzyme preparations from these two plant sources and in both cases, the enzymes acted on acceptors having a &bgr;1,2-GlcNAc residue on the Man&agr;1,3-arm, but GlcNAc-Man
5
(GlcNAc)
2
was not a good acceptor. Thus, in the processing pathway, xylose may be added after the mannosidase II step (15). However, with the xylosyltransferase from the snail,
Lymnaea stagnalis,
the biantennary oligosaccharide containing a galactose in &bgr;1,4 linkage to the GlcNAc on the Man&agr;1,3-arm was also an acceptor, although it was much less efficient (17). These studies suggested that xylose is added after the removal of the two mannoses on the Man&agr;1,6-arm, but before galactoses are added to the GlcNAc residues.
The xylose units on these N-linked oligosaccharides may play a critical role in allergenicity, and may also be important in regulating the structure of the oligosaccharide chains and the targeting of these proteins. Thus, it is important to purify this enzyme in order to study its properties and specificities in the absence of interfering activities and possible inhibitors.
The prior art is deficient in the lack of a purified &bgr;1,2-xylosyltransferase. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
In the present invention, the soybean xylosyltransferase was purified about 51,000-fold to near homogeneity, and the substrate specificities, and other properties of this enzyme were determined. Two protein bands labeled with the photoaffinity tag, azido-UDP[
32
P]-xylose, were eluted from SDS gels, transferred to PVDF membranes, and digested with Endo lys C to obtain various peptides.
In one embodiment of the present invention, there is provided an isolated and purified plant enzyme that adds a &bgr;-1,2-linked xylose to the &bgr;-linked mannose on the N-linked oligosaccharides of storage glycoproteins. Preferably, the enzyme is &bgr;1,2-xylosyltransferase. &bgr;1,2-xylosyltransferase releases peptides having the sequence selected from the group consisting of SEQ ID Nos. 1-5 upon digestion by Endo lys C. Preferably, the enzyme is obtained from the microsomal fraction of soybean cells and has a purity of about 51,000-fold to near homogeneity compared to a solubilized enzyme fraction. This &bgr;1,2-xylosyltransferase is separated on SDS gels into 56 and 59 kDa bands.
In another embodiment of the present invention, there is provided one or more oligonucleotides for cloning the gene encoding &bgr;1,2-xylosyltransferase, wherein the oligonucleotides are generated from the peptide having the sequence selected from the group consisting of SEQ ID Nos. 1-5.
In still another embodiment of the present invention, there is provided DNA encoding &bgr;1,2-xylosyltransferase selected from the group consisting of: (a) isolated DNA which encodes a xylosyltransferase; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a xylosyltransferase; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a xylosyltransferase. Preferably, the DNA has a sequence shown in SEQ ID No. 6.
In yet another embodiment of the present invention, there is provided an antibody directed against &bgr;1,2-xylosyltransferase. Preferably, this antibody can be used for localizing xylosyltransferase in a plant cell by contacting the cell with the antibody and then detecting localization of the antibody, wherein the localization of the antibody indicates the localization of the enzyme. Further, the antibody can be used for determining distribution levels of xylosyltransferase in a plant by contacting the plant with the antibody and then detecting binding activity of the antibody, wherein a higher activity indicates a higher level of xylosyltransferase in the test plant.
In still yet another embodiment of the present invention, there is provided a method for determining the effect of xylosylation of animal glycoproteins by transfecting the DNA encoding &bgr;1,2-xylosyltransferase into the animal cells.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.


REFERENCES:
Stoolmiller et al. J. Biol. Chem., 1972, vol. 247(11):3525-3532.*
Rodgers et al. Biochemical J., 1992, vol. 288(3):817-822.

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