Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-01-31
2003-01-07
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06503744
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of enzymatic synthesis of oligosaccharides, including gangliosides and ganglioside mimics.
2. Background
Gangliosides are a class of glycolipids, often found in cell membranes, that consist of three elements. One or more sialic acid residues are attached to an oligosaccharide or carbohydrate core moiety, which in turn is attached to a hydrophobic lipid (ceramide) structure which generally is embedded in the cell membrane. The ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion. Gangliosides, as well as other glycolipids and their structures in general, are discussed in, for example, Lehninger,
Biochemistry
(Worth Publishers, 1981) pp. 287-295 and
Devlin, Textbook of Biochemistry
(Wiley-Liss, 1992). Gangliosides are classified according to the number of monosaccharides in the carbohydrate moiety, as well as the number and location of sialic acid groups present in the carbohydrate moiety. Monosialogangliosides are given the designation “GM”, disialogangliosides are designated “GD”, trisialogangliosides “GT”, and tetrasialogangliosides are designated “GQ”. Gangliosides can be classified further depending on the position or positions of the sialic acid residue or residues bound. Further classification is based on the number of saccharides present in the oligosaccharide core, with the subscript “1” designating a ganglioside that has four saccharide residues (Gal-GalNAc-Gal-Glc-Ceramide), and the subscripts “2”, “3” and “4” representing trisaccharide (GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide) and monosaccharide (Gal-Ceramide) gangliosides, respectively.
Gangliosides are most abundant in the brain, particularly in nerve endings. They are believed to be present at receptor sites for neurotransmitters, including acetylcholine, and can also act as specific receptors for other biological macromolecules, including interferon, hormones, viruses, bacterial toxins, and the like. Gangliosides are have been used for treatment of nervous system disorders, including cerebral ischemic strokes. See, e.g., Mahadnik et al. (1988)
Drug Development Res.
15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz (1988)
Adv. Exp. Med. and Biol.
174: 593-600; Karpiatz et al. (1984)
Adv. Exp. Med. and Biol.
174: 489-497. Certain gangliosides are found on the surface of human hematopoietic cells (Hildebrand et al. (1972)
Biochim. Biophys. Acta
260: 272-278; Macher et al. (1981)
J. Biol. Chem.
256: 1968-1974; Dacremont et al.
Biochim. Biophys. Acta
424: 315-322; Klock et al. (1981)
Blood Cells
7: 247) which may play a role in the terminal granulocytic differentiation of these cells. Nojiri et al. (1988)
J. Biol. Chem.
263: 7443-7446. These gangliosides, referred to as the “neolacto” series, have neutral core oligosaccharide structures having the formula [Gal&bgr;-(1,4)GlcNAc&bgr;(1,3)]
n
Gal&bgr;(1,4)Glc, where n=1-4. Included among these neolacto series gangliosides are 3′-nLM
1
(NeuAc&agr;(2,3)Gal&bgr;(1,4)GlcNAc&bgr;(1,3)Gal&bgr;(1,4)-Glc&bgr;(1,1)-Ceramide) and 6′-nLM
1
(NeuAc&agr;(2,6)Gal&bgr;(1,4)GlcNAc&bgr;(1,3)Gal&bgr;(1,4)-Glc&bgr;(1,1)-Ceramide).
Ganglioside “mimics” are associated with some pathogenic organisms. For example, the core oligosaccharides of low-molecular-weight LPS of
Campylobacter jejuni
O:19 strains were shown to exhibit molecular mimicry of gangliosides. Since the late 1970s,
Campylobacter jejuni
has been recognized as an important cause of acute gastroenteritis in humans (Skirrow (1977)
Brit. Med. J.
2: 9-11). Epidemiological studies have shown that Campylobacter infections are more common in developed countries than Salmonella infections and they are also an important cause of diarrheal diseases in developing countries (Nachamkin et al. (1992)
Campylobacter jejuni: Current Status and Future Trends
. American Society for Microbiology, Washington, D.C.). In addition to causing acute gastroenteritis,
C. jejuni
infection has been implicated as a frequent antecedent to the development of Guillain-Barré syndrome, a form of neuropathy that is the most common cause of generalyzed paralysis (Ropper (1992)
N. Engl. J. Med.
326: 1130-1136). The most common
C. jejuni
serotype associated with Guillain-Barré syndrome is O:19 (Kuroki (1993)
Ann. Neurol.
33: 243-247) and this prompted detailed study of the lipopolysaccharide (LPS) structure of strains belonging to this serotype (Aspinall et al. (1994a)
Infect. Immun.
62: 2122-2125; Aspinall et al. (1994b)
Biochemistry
33: 241-249; and Aspinall et al. (1994c)
Biochemistry
33: 250-255).
Terminal oligosaccharide moieties identical to those of GD1a, GD3, GM1 and GT1a gangliosides have been found in various
C. jejuni
O:19 strains.
C. jejuni
OH4384 belongs to serotype O:19 and was isolated from a patient who developed the Guillain-Barré syndrome following a bout of diarrhea (Aspinall et al. (1994a), supra.). It was showed to possess an outer core LPS that mimics the tri-sialylated ganglioside GT1a. Molecular mimicry of host structures by the saccharide portion of LPS is considered to be a virulence factor of various mucosal pathogens which would use this strategy to evade the immune response (Moran et al. (1996a)
FEMS Immunol. Med. Microbiol.
16: 105-115; Moran et al. (1996b)
J. Endotoxin Res.
3: 521-531).
Consequently, the identification of the genes involved in LPS synthesis and the study of their regulation is of considerable interest for a better understanding of the pathogenesis mechanisms used by these bacteria. Moreover, the use of gangliosides as therapeutic reagents, as well as the study of ganglioside function, would be facilitated by convenient and efficient methods of synthesizing desired gangliosides and ganglioside mimics. A combined enzymatic and chemical approach to synthesis of 3′-nML
1
and 6′-nLM
1
has been described (Gaudino and Paulson (1994)
J. Am. Chem. Soc.
116: 1149-1150). However, previously available enzymatic methods for ganglioside synthesis suffer from difficulties in efficiently producing enzymes in sufficient quantities, at a sufficiently low cost, for practical large-scale ganglioside synthesis. Thus, a need exists for new enzymes involved in ganglioside synthesis that are amenable to large-scale production. A need also exists for more efficient methods for synthesizing gangliosides. The present invention fulfills these and other needs.
REFERENCES:
patent: 5352670 (1994-10-01), Venot et al.
patent: 5374541 (1994-12-01), Wong et al.
patent: 5545553 (1996-08-01), Gotschlich
patent: WO92/16640 (1992-10-01), None
patent: WO96/32491 (1996-10-01), None
Aspinall et al., “Lipopolysaccharides fromCampylobacter jejuniAssociated with Guillain-Barre Syndrome Patients Mimic Human Gangliosides in Stucture”Infect. Immun., 62: 2122-2125 (1994a).
Aspinall et al., “Lipopolysaccharides ofCampylobacter jejuniSerotype O:19:Structures of Core Oligosaccharide Regions from the Serostrain and Two Bacterial Isolates from Patients with the Guillain-Barre Syndrome”Biochemistry, 33: 241-249 (1994b).
Aspinall et al., “Lipopolysaccharides ofCampylobacter jejuniSerotype O:19: Antigen Chains from the Serostrain and Two Bacterial Isolates from Patients with the Guillain-Barre Syndrome”Biochemistry, 33: 250-255 (1994c).
Belunis et al., “Biosynthesis of Endotoxin”J. Biol. Chem., 267: 9988-9997 (1992).
Gaudino et al., “A Novel and Efficient Synthesis of Neolacto Series Gangliosides 3′-nLM1and 6′-nLM1”J. Am. Chem. Soc., 116: 1149-1150 (1994).
Gilbert et al., “Cloning of the Lipooligosaccharide &agr;-2,3-Sialytransferase from the Bacterial PathogensNeisseria meningitidisandNeisseria gonorrhoeae” J. Biol. Chem., 271: 28271-28276 (1996).
Ito et al., “Synthesis of Bioactive Sialosides”Pure Appl. Chem., 65: 753 (1993).
Kuroki, “Campylobacter jejuniStrains from Patients with Guillain-Barre Syndrome Belong Mostly to Penner Serogroup 19 Contain &bgr;-N-Acetylglucosamine Residues”Ann. Neurol., 33: 243-247 (1993).
Gilbert Michel
Wakarchuk Warren W.
National Research Council of Canada
Prouty Rebecca E.
Rao Manjunath N.
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