&bgr;1-4 N-acetylglucosaminyltransferase and gene encoding

Details &bgr;1-4 N-acetylglucosaminyltransferase and gene encoding &bgr;1-4 N-acetylglucosaminyltransferase and gene encoding

1998-08-12

2002-01-15

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S193000, C435S320100, C435S325000, C435S252300, C536S023200

Reexamination Certificate

active

06338955

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a novel N-acetylglucosaminyl-transferase (GlCNAc transferase) which recognizes a specific sugar chain structure in a saccharide and introduces thereinto a GlcNAc &bgr;1→4 branching structure.
BACKGROUND ART
1. Glycoproteins
Most of proteins occurring in nature are not simple proteins composed of amino acids alone, but “mature” proteins having sugar chains and other substances such as phosphates and lipids attached thereto. Therefore, the development of simple protein-type products produced by
Escherichia coli
as a host has involved various problems because such products lack the maturing process of proteins. Since all of secretion-type physiologically active proteins (e.g. cytokines) are glycoproteins with a few exceptions, the function and the role of sugar chains have attracted attention as the most important point in the development of biological pharmaceuticals.
Sugar chains in glycoproteins are classified roughly into Asn-linked type, mucin-type, O-linked GlcNAc type, GPI anchor type and proteoglycan type [Makoto Takeuchi, “Glycobiology Series 5: Glycotechnology”, Kihata, Hakomori and Nagai (eds.), Kodansha Scientific Co., (1994), 191-208]. Each of these types of sugar chains has its own biosynthesis pathway and a discrete physiological function. Asn-linked sugar chains are distributed widely in molds, yeasts, insects, plants and animals. The basic biosynthesis pathway for Asn-linked sugar chains is conserved beyond species (FIG.
1
). A sugar chain(s) characteristic of a specific species is(are) formed on the outer side (called the “non-reducing terminal side”) of the core sugar chain moiety which is common in the biosynthesis of Asn-linked sugar chains. A mannan-type sugar chain in whicha &agr; 1,3- and &agr; 1,2-branching mannose residues attach to a main chain extending via &agr; 1,6 linkages is a sugar chain structure characteristic of fungi such as yeasts (see Panel a in
FIG. 2
) [Hiroshi Nakajima, Sugar Chain Technology, Industry Survey Association (1992), 384-397]. On the other hand, in insects, plants and animals., extension of mannose residues is not observed; instead, a high mannose type sugar chain is formed which is a sugar chain transferred from a dolichol intermediate and only trimmed (see Panel c in FIG.
2
). A unique structure having characteristic xylose or the like (see Panel b in
FIG. 2
) is also observed in insects, plants and mollusks. In animals, characteristic sugar chain structures such as complex type sugar chain (Panel e in
FIG. 2
) and hybrid type sugar chain (Panel d in
FIG. 2
) are observed; in the former, GlcNAc branching structures are formed in a once trimmed sugar chain, and addition of other kinds of monosaccharides such as galactose and sialic acid forms complicated structures; in the latter, both a complex type sugar chain and a high mannose type sugar chain are present [Kiyoshi Furukawa, Sugar Chain Technology, Industry Survey Association (1992), 64-75].
Such sugar chains as described above are conferred on most of cell surface proteins and secretion proteins, and are thought to play important roles which determine the natures and properties of cells and proteins. Among all, the portion of a sugar chain structure which forms a branch elongating like antennas from the common core sugar chain is called a sugar chain branching structure. This structure is believed to have a function to give an organism recognition ligand (i.e., the end portion of the sugar chain) a high degree of freedom to thereby provide chances for multipoint recognition and another function to maximize the protection ability for the protein moiety by greatly increasing the space-occupying volume (Takeuchi et al., supra). Therefore, by controlling the branching structure of sugar chains, it is possible to modify the physiological functions, sush as itn vivo stability, in vivo kinetics and organ-targeting properties of glycoproteins in various ways. In view of this, technology to control branching structures of sugar chains is expected as biotechnology of the next generation for the development of glycoprotein-type pharmaceuticals which are “tender to humans”.
2. Physiological Significance of Glycoprotein Sugar Chains
Sugar chains of secretion type glycoproteins exhibit excellent functions in biosynthesis, intracellular sorting, masking of antigenicity, in vivo stability and organ-targeting properties of glycoproteins. Sugar chains of cell surface proteins are known to change in response to changes in cells (such as differentiation, change to a morbid state, canceration). In particular, it has been reported that there is a close relation between the metastasis of cancer and the branching structure of sugar chains.
(1) Masking of Antigenicity
It is considered that sugar chains have a high degree of freedom in terms of steric structure and thus are moving freely like propellers. Therefore, protein molecules such as proteases and antibodies against proteins not having affinity to sugar chains are shook off by the sugar chains and thus cannot gain access to the protein moiety. As a result, even if there is antigenicity in the peptide moiety near the sugar chain binding site, antibody molecules cannot have access to the peptide moiety. Thus, an antigen-antibody reaction is extremely difficult to occur. Further, when a glycoprotein has been captured by a macrophage and the degradation products are presented as antigen, receptors are difficult of access to the peptides around the sugar chain binding site. Thus, antigenic stimulation is difficult to occur. Actually, it is reported that when sugar chains have been introduced into the central portion of the antigenic peptide of ovalbumin lysozyme, the binding of MHC class II molecules to the antigen is remarkably inhibited [Mouritsen, S., Meldal, M., Christiansen-Brams, I., Elsner, H. and Werdelin, O., Eur. J. Immunol., (1994), 24, 1066-1072]. The effect of such masking of antigenicity becomes greater as the volume occupied by sugar chains is greater. Thus, it is considered that the development of a branching structure contributes to the effect of such masking greatly.
(2) In Vivo Stability
With respect to erythropoietin which is the first glycoprotein-type pharmaceutical ever produced from a transgenic animal cell as a host, the functions of sugar chains thereof have been studied thoroughly. As a result, it has been shown that the sugar chains of erythropoietin work inhibitorily against the binding of erythropoietin with its receptor but make a decisive contribution to the retaining of the active structure and the improvement of in vivo kinetics; as a whole, the sugar chains have been shown to be essential for expression of the pharmacological activity of erythropoietin (Takeuchi, M. and Kobata, A., Glycobiology (1991), 1, 337-346). In particular, a strong correlation between the number of antennae in sugar chains and the pharmacological effect of erythropoietin has been found, and thus the importance of its branching structure (a branching structure formed by GlcNAc residues attaching to the core sugar chain) which never attracted attention has been made clear for the first time [Takeuchi, M., Inoue, N., Strickland, T. W., Kobata, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A., Proc. Natl. Acad. Sci. USA, (1989), 86, 7819-22]. The major cause of the above phenomenon is explained as follows: erythropoietin without developed branching structure is cleared rather rapidly in kidney and, as a result, the in vivo residence time of such erythropoietin becomes shorter [Misaizu, T., Matsuki, S., Strickland, T. W., Takeuchi, M., Kobata, A. and Takasaki, S., Blood, (1995), 86, 4097-4104].
(3) Organ Targeting Property
Most of biological tissues have lectin-like receptors and use then in cell-cell interactions or to uptake glycoproteins from blood. The asialoprotein-binding lectin in liver is a representative example of a clearance system for aged glycoproteins [Toshihiro Kawasaki,

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