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Glycosylation revisited
Trends in Biotechnology, Vol. 3, No. 9, 1985
218
Drosophila chromosomes. This strategy has been used to integrate into the Drosophila genome DNA coding for the Epstein-Barr virus nuclear antigen, the expression of which is normally limited to human lymphoid tissue. Visualization with antibodies
revealed that viral nuclear antigen is transported to the nucleus, suggesting that synthesis of this protein need not be restricted to a specific human cell type, and that in insect ceils processing is fundamentally like that in mammalian cells.
Glycosylation revisited Among the many post-translational modifications of proteins, glycosylation remains somewhat mysterious. Not only has it been impossible to ascribe a unique function to the carbohydrate moieties, but it seems that each glycosylated protein must be evaluated individually to determine the importance of glycosylation to its function and stability. This assessment is an important economic consideration in the choice of a host organism for the production ofa glycoprotein as a pharmaceutical product. The subject has been reviewed in this journal earlier this year 1and a recent article by Sairam and Bhargavi2, which describes the role of carbohydrates in fertility hormones, throws additional light on the subject. Many proteins anchored on, or secreted by, eukaryotic cells are glycosylated. However, most prokaryotic, cells do not produce glycoproteins. Although the functions ofglycosylation are not entirely predictable, general differences between deglycosylated molecules and their natural counterparts have been defined. Among these differences, a few of biological interest can be mentioned. Antigenicity: carbohydrates can be part of an epitope. Their absence or removal from glycoproteins can unmask additional antigenic determinants on the peptide chain. Their effect on antigenicity can also be detected at a remote location on the molecule inasmuch as glycosylation affects the folding of the chain. Stability: glycosylation can protect against proteolytic enzymes in actual physiological use as well as during recovery and purification. Solubility: the contribution of negative charges by terminal sialic acid groups on the carbohydrate moieties can be significant in the solubilization and, therefore, in the activity of glycopro-
teins at physiological concentrations. Tertiary structure: additional negative charges can significantly alter the conformation of the molecule. Also, there is evidence that early glycosylation events during translation influence folding of the chain. It is logical to expect that conformational changes will affect the activity ofglycoproteins.
ANN M. FALLON
Department of Microbiology~ UMDNJSchool of Osteopathic Medicine, Piscataway, NJ 08854, USA.
and JUDITH H. WILLIS Department of Genetics and Development, University of Illinois, Urbana, IL 61801, USA.
a subunit. The nature of the carbohydrate moiety of a glycoprotein and the extent to which a protein is glyco. sylated depend on which host is used. It is not yet clear whether such differences will affect the biological activity of a given protein. From a biotechnological point of view, the finding is important because it is the first time that a specific functional role for the carbohydrate moiety of a glycoprotein has been assigned: no functional difference attributable to glycosylation has been detected when the unglycosylated and glycosylated forms of other biologically active proteins, such as a-interferon, have been compared. It may, therefore, be necessary to seek eukaryotic host systems for producing some glycoproteins. The a-chain could also be an excellent model of glycosylation in different eukaryotic non-mammalian host systems. This is being pursued in several laboratories 3-5. Such systems include yeasts and insect cells which could be less expensive to grow than mammalian cells. This might be useful in producing glycoproteins assuming that the glycosylation pattern is similar to that in the natural producing cell.
The recent work by Sairam and Bhargavi confirms the link between glycosylation and functional activity of gonadotropic hormones 2. These hormones, which include choriogonadotropin, follicle-stimulating hormone and luteinizing hormone, all have two non-covalently linked subunits, a and /3, both of which are characterized by a high carbohydrate content. The gonadotropic hormones share the same a chain within an animal species, and their structure is highly conserved among mammals. The hormones bind to receptor sites on a target cell and the complex activates adenylate cyclase (thereby regulating cellular metabolism). The subunits can be isolated and recombined to form a product identical to the native hormone. If the/3 chain is deglycosylated, re- References combination with a normal a chain 1 Berman, P. W. and Lasky, L. A. (1985) yields a product with normal receptor Trends BioteehnoL 3, 51-53 binding and adenylate cyclase activa2 Sairam, M. R. and Bhargavi, ( N. (1985) Science 229, 65-67 tion. In the reverse case, the product obtained by recombining a normal fl 3 Wood,C. R., Boss,M. A., Kenten, J. H., Calvert, J. E. and Roberts, N. A. (1985) subunit with a deglycosylated a subNature 314, 446-449 unit binds as well, or better, to the 4 Innis, M. A., Holland, M, J., McCabe, receptor site. However, the complex P. C., Cole, G. E., Whittman, V. P., Tal, R., Watt, K. W. K., Gelfand, D. H., cannot activate adenylate cyclase. Holland, J. P. and Meade, J. H. (1985) Therefore, the carbohydrate moiety is Science 228, 21-26 directly or indirectly involved in the 5 Summers,M. (1983) as quoted in Wall transduction of the biological signal. Street Journal 3 Star, (Eastern Edn, As noted by the authors, this fending Princeton, NJ) 8 December 1983, p. 47 may be important in understanding JACQUESJ. DELENTE the mode of action of glycoprotein hormones and may be of evolutionary significance considering the high Chembiotech, 6808 Renita Lane, Bethesda, degree of structural conservation of the MD 20817, USA.
© 1985,ElsevierSciencePublishersB.V.,Amsterdam 0166-9430/85/$02.00
218
Drosophila chromosomes. This strategy has been used to integrate into the Drosophila genome DNA coding for the Epstein-Barr virus nuclear antigen, the expression of which is normally limited to human lymphoid tissue. Visualization with antibodies
revealed that viral nuclear antigen is transported to the nucleus, suggesting that synthesis of this protein need not be restricted to a specific human cell type, and that in insect ceils processing is fundamentally like that in mammalian cells.
Glycosylation revisited Among the many post-translational modifications of proteins, glycosylation remains somewhat mysterious. Not only has it been impossible to ascribe a unique function to the carbohydrate moieties, but it seems that each glycosylated protein must be evaluated individually to determine the importance of glycosylation to its function and stability. This assessment is an important economic consideration in the choice of a host organism for the production ofa glycoprotein as a pharmaceutical product. The subject has been reviewed in this journal earlier this year 1and a recent article by Sairam and Bhargavi2, which describes the role of carbohydrates in fertility hormones, throws additional light on the subject. Many proteins anchored on, or secreted by, eukaryotic cells are glycosylated. However, most prokaryotic, cells do not produce glycoproteins. Although the functions ofglycosylation are not entirely predictable, general differences between deglycosylated molecules and their natural counterparts have been defined. Among these differences, a few of biological interest can be mentioned. Antigenicity: carbohydrates can be part of an epitope. Their absence or removal from glycoproteins can unmask additional antigenic determinants on the peptide chain. Their effect on antigenicity can also be detected at a remote location on the molecule inasmuch as glycosylation affects the folding of the chain. Stability: glycosylation can protect against proteolytic enzymes in actual physiological use as well as during recovery and purification. Solubility: the contribution of negative charges by terminal sialic acid groups on the carbohydrate moieties can be significant in the solubilization and, therefore, in the activity of glycopro-
teins at physiological concentrations. Tertiary structure: additional negative charges can significantly alter the conformation of the molecule. Also, there is evidence that early glycosylation events during translation influence folding of the chain. It is logical to expect that conformational changes will affect the activity ofglycoproteins.
ANN M. FALLON
Department of Microbiology~ UMDNJSchool of Osteopathic Medicine, Piscataway, NJ 08854, USA.
and JUDITH H. WILLIS Department of Genetics and Development, University of Illinois, Urbana, IL 61801, USA.
a subunit. The nature of the carbohydrate moiety of a glycoprotein and the extent to which a protein is glyco. sylated depend on which host is used. It is not yet clear whether such differences will affect the biological activity of a given protein. From a biotechnological point of view, the finding is important because it is the first time that a specific functional role for the carbohydrate moiety of a glycoprotein has been assigned: no functional difference attributable to glycosylation has been detected when the unglycosylated and glycosylated forms of other biologically active proteins, such as a-interferon, have been compared. It may, therefore, be necessary to seek eukaryotic host systems for producing some glycoproteins. The a-chain could also be an excellent model of glycosylation in different eukaryotic non-mammalian host systems. This is being pursued in several laboratories 3-5. Such systems include yeasts and insect cells which could be less expensive to grow than mammalian cells. This might be useful in producing glycoproteins assuming that the glycosylation pattern is similar to that in the natural producing cell.
The recent work by Sairam and Bhargavi confirms the link between glycosylation and functional activity of gonadotropic hormones 2. These hormones, which include choriogonadotropin, follicle-stimulating hormone and luteinizing hormone, all have two non-covalently linked subunits, a and /3, both of which are characterized by a high carbohydrate content. The gonadotropic hormones share the same a chain within an animal species, and their structure is highly conserved among mammals. The hormones bind to receptor sites on a target cell and the complex activates adenylate cyclase (thereby regulating cellular metabolism). The subunits can be isolated and recombined to form a product identical to the native hormone. If the/3 chain is deglycosylated, re- References combination with a normal a chain 1 Berman, P. W. and Lasky, L. A. (1985) yields a product with normal receptor Trends BioteehnoL 3, 51-53 binding and adenylate cyclase activa2 Sairam, M. R. and Bhargavi, ( N. (1985) Science 229, 65-67 tion. In the reverse case, the product obtained by recombining a normal fl 3 Wood,C. R., Boss,M. A., Kenten, J. H., Calvert, J. E. and Roberts, N. A. (1985) subunit with a deglycosylated a subNature 314, 446-449 unit binds as well, or better, to the 4 Innis, M. A., Holland, M, J., McCabe, receptor site. However, the complex P. C., Cole, G. E., Whittman, V. P., Tal, R., Watt, K. W. K., Gelfand, D. H., cannot activate adenylate cyclase. Holland, J. P. and Meade, J. H. (1985) Therefore, the carbohydrate moiety is Science 228, 21-26 directly or indirectly involved in the 5 Summers,M. (1983) as quoted in Wall transduction of the biological signal. Street Journal 3 Star, (Eastern Edn, As noted by the authors, this fending Princeton, NJ) 8 December 1983, p. 47 may be important in understanding JACQUESJ. DELENTE the mode of action of glycoprotein hormones and may be of evolutionary significance considering the high Chembiotech, 6808 Renita Lane, Bethesda, degree of structural conservation of the MD 20817, USA.
© 1985,ElsevierSciencePublishersB.V.,Amsterdam 0166-9430/85/$02.00