Many proteins associated with the external surfaces of cell membranes or actively secreted from cells are commonly modified by the addition of one or more carbohydrate units to the side chains of particular amino acids [R. D. Marshall, Ann. Rev. Biochem., 41, pp. 673–702 (1972)]. Such proteins, known as glycoproteins, display the properties of proteins in general, as well as properties typical of the attached carbohydrate. The carbohydrate monomers typically attached to glycoproteins include galactose, mannose, glucose, N-acetylglucosamine, N-acetylgalactosamine, fucose, xylose, sialic acid and others. The carbohydrate units are usually attached through the hydroxyl groups of serine and threonine side chains, or the amide nitrogen atom of asparagine side chains. The carbohydrate side chains are arranged in a variety of chain lengths and branching patterns [P. V. Wagh and O. P. Bahl, Crit. Rev. Biochem., 10, pp. 307–77 (1981)].
Glycoproteins exhibit a range of protein functions, including catalysis of chemical transformations, proteolysis of proteins, binding of ligands and transport of ligands to and across membranes. Additionally, glycoproteins frequently perform functions associated with cellular communication, including protein-protein recognition, protein-carbohydrate recognition, protein-DNA recognition, pathogen recognition by antibodies, antigen presentation by CD4 and CD8 membrane glycoproteins, and targeting of proteins to specific locations.
Glucose oxidase exemplifies an enzyme glycoprotein that catalyzes the oxidation of β-D-glucose to D-glucono-1,5-lactone. The reaction consumes one mole of oxygen and produces one mole of hydrogen peroxide per mole of glucose. The active glycoprotein glucose oxidase, or β-D-glucose:oxygen 1-oxidoreductase [enzyme commission number 1.1.3.4] forms a dimer with a molecular weight of 150–180 kDa. Each monomer consists of 583 amino acids residues, one co-factor molecule of flavin adenine dinucleotide (FAD) and has a carbohydrate content of approximately 16% by weight. The three dimensional structure of the deglycosylated protein has been determined by X-ray crystallography [H. J. Hecht, H. M. Kalisz, J. Hendle, R. D. Schmid and D. Schomburg, J. Mol. Biol., 229, pp. 153–72 (1993)]. Due to the importance of the quantitative determination of glucose in medicine and industry, glucose oxidase is considered a prime candidate for the development of biosensors [H. J. Hecht, D. Schomburg, H. M. Kalisz and R. D. Schmid, Biosensors and Bioelectronics, 8, pp. 197–203 (1993)]. Glucose oxidase may be advantageously used in the food, drug and cosmetics industries, because of its ability to interconvert oxygen and hydrogen peroxide. Commercially available glucose oxidase is usually isolated from Aspergillus niger. 
The stereoselectivity and specific activity of enzymatic glycoproteins may be exploited for use in industrial syntheses. For example, glutaraldehyde crosslinked crystals of Lipase from Candida rugosa may be used to synthesize optically pure compounds [J. J. Lalonde, C. Govardhan, N. Khalaf, A. G. Martinez, K. Visuri and A. L. Margolin, J. Am. Chem. Soc., 117, (26) pp. 6845–52, (1995)].
In infectious diseases, glycoproteins are involved in the initiation and maintenance of infection, as well as host humoral and cellular immune responses against infection. The surface proteins of many viruses are glycoproteins. Examples of such glycoproteins include, for example, gp120 and gp41 of the Human Immunodeficiency Virus (HIV), which causes AIDS [H. Geyer, C. Holschbach, G. Hunsmann and J. Schneider, J. Biol. Chem., 263 (24), pp. 11760–67, (1988)] and the hemagglutinin (HA) and neuraminidase (NA) of Influenza Virus, which causes Flu.
Viral receptors, the cellular proteins recognized by invading viruses, are found on the surface of cells and therefore are frequently either glycoproteins or carbohydrates. The CD4 molecule, which is recognized by HIV-1 gp120, is a glycoprotein. Similarly, Influenza virus hemagglutinin binds to terminal N-acetylneuraminic acid residues of sialoglycoproteins and enters the cell through receptor mediated endocytosis [J. White, M. Kielian and A. Helenius, Quart. Rev. of Biophys., 16, pp. 151–95, (1983)].
In spite of the tremendous medical, chemical, pharmaceutical and industrial potential of glycoproteins, their development has, in many instances, lagged far behind that of unglycosylated proteins.
As compared with unglycosylated proteins, the frequent association of glycoproteins with biological membranes and other membrane proteins, render glycoproteins significantly more difficult to purify and utilize for medical and industrial processes. The use of glycoproteins faces additional barriers because relatively little is known about their three-dimensional structure and the requirements for stabilization when faced with harsh environments. However, due to the specialized functions of glycoproteins, many benefits can be realized by overcoming the barriers to widespread large scale use of glycoproteins in industrial, chemical and medical applications.
One unique approach to overcoming barriers to the widespread use of proteins generally is crosslinked enzyme crystal (“CLEC™”) technology [N. L. St. Clair and M. A. Navia, J. Am. Chem. Soc., 114, pp. 4314–16 (1992)]. Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases and other protein digestion agents, high temperature or extreme pH and organic solvents. In such environments, crosslinked enzyme crystals remain insoluble and stable.
One physical result of “CLEC™” technology is that the surface exposed amino acid side chains of the protein, in the crystal lattice, are covalently modified with the crosslinking agents. This modification stabilizes the crystal lattice, at the same time altering elements of the surface structure to gain the benefits of stabilization. For most applications involving protein crystals, any potential limitation resulting from minor surface modifications is overcome by the gains in stability achieved in the crystals. However, applications involving vaccines, and immunotherapeutics using glycoproteins, often require that the surface exposed protein structures, known as epitopes, precisely maintain the original structure. This may require stabilization without chemical modification of the amino acid side chains or perhaps with minor levels of chemical modification of the amino acid side chains.
USPTO Backgrounds
USPTO Backgrounds