N-glycosylation and the production of recombinant glycoproteins

TIBTECH

-

MAY 1989 [Vol. 7]

Characteristics of N-glycosylation Three important characteristics of polypeptide N-glycosylation relevant to this article are that a single glycoprotein carries several different oligosaccharides, that both the amino acid sequence and the cell type influence N-glycosylation and that, under stable conditions, N-glycosylation is highly reproducible.

N-glycosylation and the production of recombinant glycoproteins Raj B. Parekh, Raymond A. Dwek, Christopher J. Edge and Thomas W. Rademacher Many polypeptides of therapeutic and diagnostic interest that are being produced in recombinant form for in-vivo administration carry, in their native form, N-linked oligosaccharides. In this article we review certain biosynthetic aspects and functional consequences of N-glycosylation that are relevant to the production and administration of recombinant glycoproteins. In so doing, we hope to increase awareness of the biological significance of N-linked oligosaccharides, and to emphasize the need to consider the N-glycosylation of a recombinant glycoprotein prior to therapeutic administration. The secreted and cell surfaceassociated polypeptides of higher organisms are subject to a variety of post-translational modifications involving carbohydrate moieties (Fig. 1). Of these, one of the most common is N-glycosylation, the attachment of oligosaccharides to the polypeptide through an N-glycosidic bond 1'2. N-glycosylation occurs in a wide variety of species throughout the eukaryotic kingdom, and generally involves attachment of oligosaccharide to the asparagine of Asn-X-Ser/Thr. Despite the ubiquity of N-glycosylation and its presence through evolution, analysis of the structure and function of N-linked oligosaccharides has, until recently, lagged considerably behind our understanding of polypeptides, nucleic acids and lipids. There are several reasons for this. Firstly, there are several different oligosaccharide R. B. Parekh is at Oxford GlycoSystems Ltd, Unit 4, Hitching Court, B]acklands Way, Abingdon Business Park, Oxon 0X14 1RG, UK, and R. A. Dwek, C. J. Edge and T. W. Rademacher are at the Oxford Glycobiology Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK.

structures at each N-glycosylation site. Secondly, there has been the problem that release of all oligosaccharide structures from a polypeptide has been selective: the use of N-glycanase to release the oligosaccharide may bias the types of structure released. Subsequent fractionation of the complex oligosaccharide mixture that is usually obtained can also be difficult. Thirdly, there is no single and simple technique available for sequence analysis of the relatively small amounts of highly branched purified oligosaccharides that are usually obtained. Many of these difficulties are being overcome 3'4, and it is becoming clear that N-linked oligosaccharides are highly branched, structurally complex molecules possessing secondary and tertiary structure, able to undergo precise conformational transitions 5. While the structural data on glycoproteins have tended to focus principally on the structure of the polypeptide, the contribution of Nlinked oligosaccharides to the structure 6, and to the overall threedimensional surface presented by a glycoprotein is quite considerable (Fig. 2).

Diversity An individual N-glycosylation site is usually associated with several oligosaccharides 7, and most glycoproteins have two or more Nglycosylation sites. Therefore, a glycoprotein really exists as different sets of molecules that all share a common polypeptide but differ in the number, location or structure of Nlinked oligosaccharides (Fig. 3). By creating these different molecules, or glycoforms, of a polypeptide, Nglycosylation diversifies a polypeptide structurally; in some cases at least, this also-leads to functional diversity 8. For example, tissue-type plasminogen activator (tPA) can be separated into subsets of glycoforms, with each subset exhibiting unique kinetics of fibrin-dependent plasminogen activation 9. Amino acid sequence and ceil-type effects Both the amino acid sequence (or protein 'matrix') and the cell type play an important part in oligosaccharide processing. The biosynthesis of N-linked oligosaccharides has been elucidated 1°, but the details of the control of N-glycosylation are not understood. While the polypeptide clearly does affect its own capacity for N-glycosylation, so too does the cell type. Not all cells or tissues perform N-glycosylation in the same way. Often a single polypeptide expressed in two different cell types or tissues of the same or different species is N-glycosylated differently. For example, the two forms of the rat T-cell marker Thy-1 (Ref. 7) recovered from brain or thymocytes are N-glycosylated so differently that they do not have a single glycoprotein molecule in common (Fig. 3), even though they have identical polypeptide sequences and probably similar polypeptide tertiary

(~) 1989, Elsevier Science Publishers Ltd (UK)

0167 - 9430/89/$02.00

TIBTECH - MAY 1989 [Vol. 7]

Fig. 1

structures (the immunoglobulin fold). However, Asn23 in both forms was associated principally with oligomannose-type structures and Ash74 with complex-type structures, indicating the way in which the polypeptide influences its own Nglycosylation. N-glycosylation is species-specific 3. The non-reducing terminal Gal(~I3)GlcNAc residue, for example, is characteristic of serum glycoproteins of bovine and rat species, but not of humans, where Gal(~I-4)GlcNAc predominates. Furthermore, oligosaccharide determinants can define barriers to inter-species exchange of glycoproteins: there are naturally circulating antibodies against certain oligosaccharide determinants that are not found on the circulating glycoproteins of the hosts but are prevalent in other species 11'12. A particularly good example of this is the Gal(~l-3)Gal determinant: there is an inverse relationship between the presence of this determinant on glycoproteins and the incidence of antibodies against it in serum 11.

l

,-

:,

....

, {ST ;I

T,rl 0

II

0 II

vv-£-EfN--O--p-- 0I O~o-I~osifol

0 The major post-translational modifications involving carboI hydrate of the secreted or cell-surface polypeptides of higher -0-P=0 organisms. N-glycosylation involves the covalent attachment I 0 of an oligosaccharide moiety to the NH2 groups of asparagine in Asn-X-Ser/Thr triplets via an N-glycosidic bond. N-linked oligosaccharides are generally located at 13-turns and most O0 glycoproteins carry two or more N-linked oligosaccharides~'2 II O=C C=O O-glycosylation involves the covalent attachment of an oligosaccharide moiety to hydroxyl groups of serine (or threonine) of the polypeptide via an O-glycosidic bond 7. In the covalent attachment of a polypeptide via a glycosylphosphatidylinositol (GPI) membrane anchor, the C-terminal amino acid residue is attached to a glycan moiety through an ethanolamine phosphate bridge; the glycan moiety is attached to diacylglycerol through an inositol phosphate bridge 36 An increasing number of cell surface glycoproteins have been found to be anchored into the lipid bilayer via such a group.

-- Fig. 2

Physiological effects The N-glycosylation of a given site during a stable and constant physiological condition is conserved and highly reproducible13, but changes in physiological condition can induce changes in N-glycosylation 14. Whether N-glycosylation is affected by culture conditions such as oxygen tension and cell density has not yet been fully established. In short, the final product of a eukaryotic gene is a set of glycoforms of that polypeptide. The structure of each glycoform and the overall composition of the set of glycoforms depends on the polypeptide structure, the cell (or tissue) type (both species of origin and state of differentiation), and in some cases the physiological condition of the cellular population or tissue. Production of recombinant glycoproteins Many polypeptides of therapeutic or diagnostic importance (e.g. erythropoietin, tPA and factor VIII) are N-glycosylated. A common strategy for isolating significant amounts of a polypeptide is to express the

A computer-generated model of one of the glycoforms of rat Thy-1 from the thymus. The sites of attachment for oligomannose (a), complex (sialylated lactosaminoglycan, b) and sialylated biantennary (c) oligosaccharides, and the site of attachment of the GPI anchor (d) are shown. Rat Thy-1 is part of the immunoglobulin superfamily so the peptide tertiary structure was generated using the crystal structure of the immunoglobulin variable region Fab New 37. Individual amino acid residues were changed to give the correct primary sequence of Thy-1 whilst retaining the overall tertiary structure of Fab New. The new peptide was then energy-minimized using the AMBER program 38 to give this structure. The appropriate oligosaccharides were then attached to the correct positions on the polypeptide and placed in time-averaged configurations given by NMR and energy minimization.

TIBTECH- MAY 1989[Vol. 7] - - Fig. 3

Occurrence (%) ThymocyteThy-1

Brain Thy-1 Occurrence(%) 98

70

L+ 30

NH2

23

7/.

t7/,~

1

NH2 44

98

-~

NH2

f'~

47

- ~23

~

NH2

6

NH2 N H 2 ~

The "composite" glycoforms of rat brain and thymocyte Thy-l. Symbols represent the types of structure presL- I 8 NH2 ~ ent at each site (see Ref. 7 for details). The structures of the composite glycoforms 8 NH 2 ~-----are deduced from structural analysis of the oligosaccharides of each N-glycoNH 2 sylation site of each form of Thy-1. Since there is no composite glycoform in common between rat brain and thymocyte Thy-1, there can be no Thy-1 glycoprotein molecule in common between these two tissues.

12

NH2~--

recombinant DNA encoding the polypeptide from one species in a cell line derived from another species. For example, most human polypeptides of therapeutic interest have been expressed in the Chinese hamster ovary (CHO) cell line. Given the influence of the cell type, one would not expect the recombinant glycoprotein recovered to have the same set of glycoforms as the native form. Detailed comparisons of the native and recombinant forms have been made for three different human polypeptides, namely human interferon-~ (Ref. 15), human erythropoietin 16'17 and human tPA (R. B. Parekh e t a ] . , unpublished). The native forms were never N-glycosylated in the same way as any of the recombinant forms. For example, human interferon-J31 produced in mouse epithelial cells (C127) and a human lung adenocarcinoma line (PC8) carried oligosaccharide structures not present in the native human interferon ~1 (Ref. 15). Interferon ~ produced in CHO cells carried a small proportion (5%) of oligosaccharides not found on the native form, lacked some oligosaccharides found on the native form and carried common ones in different relative amounts ~5, suggesting major changes in the relative

incidence of individual glycoforms. The results for human erythropoietin were similar. The recombinant form produced in baby hamster kidney (BHK) cells 16 carried oligosaccharides not found on human urinary erythropoietin, while in CHO cells ~r the oligosaccharides were similar to those on native erythropoietin, but present in quite different relative amounts. In the case of tPA (R. B. Parekh et al., unpublished), both the recombinant form from CHO cells, and that from a murine (C127) cell line carried oligosaccharides not found on a native human tPA, as did the tPA expressed by the human Bowes melanoma cell line. These three examples highlight two important points in assessing the N-glycosylation of a recombinant glycoprotein. Firstly, it is difficult to isolate and, in some cases even to define, the 'native' form of a glycoprotein. For example, the 'native' interferon ~1 (Ref. 15) was isolated from foreskin fibroblasts after poly(I).poly(C) induction, the erythropoietin a6'17 was isolated from the urine of patients with aplastic anaemia, and the tPA (R. B. Parekh et al., unpublished) from cultured human colon fibroblasts. Furthermore, changes in polypeptide Nglycosylation can occur naturally in

response to changes in physiological condition, leading to the production of natural variants of a polypeptide of different biological activity TM. Secondly, the native and recombinant forms of a polypeptide will almost certainly differ in their Nglycosylation, either in the types of oligosaccharide present or in their relative distribution. Given both the diversity of glycoforms of a particular protein and the variation in the 'native' forms of glycoproteins, how significant are the differences between native glycoproteins and their recombinant counterparts? Are glycoprotein functions affected and, if so, what are the consequences for the pharmaceutical use of recombinant glycoproteins?

Functions of N-linked oligosaccharides In many cases, the N-linked oligosaccharides of a polypeptide contribute to and are an intrinsic part of the physiological activity of that glycoprotein. Many glycoproteins are multifunctional and engage in several intermolecular interactions. Each function of the glycoprotein may be derived to differing extents from different glycoforms, and the 'composite' activity of the glycoprotein in a particular function wilt reflect a weighted average of the activity and incidence of each glycoform. Not only do oligosaccharides affect the physical properties of a glycoprotein I~, but they can also influence its bioactivity, bio-distribution, immunogenicity and circulatory lifetime 14'2°. There are many examples of altered bioactivity. Coupling of the activation of adenylate cyclase to receptor occupancy by the gouadotropic hormones requires specific N-linked oligosaccharides on the hormone e~-subunit 21. In human chorionic gonadotropin, the oligosaccharides of the ~-subunit are needed for efficient secretion and assembly of the g]ycohormone dimers 22. The bioactivity of thyroid stimulating hormone can be naturally altered by changes in N-glycosylation 18. Similarly, changes in N-glycosylation can convert IgEbinding protein into an enhancer or a suppressor of IgE biosynthesis 23. Regional differences in agonist and

TIBTECH- MAY 1989 [Vol. 7]

antagonist binding to the brain benzodiazepine receptor have been attributed to differences in N-glycosylafion 24. The binding properties of specific cell-surface hormone receptors can be selectively controlled through changes in receptor Nglycosylation 25. Moreover, changes in N-glycosylation may be of pathological significance in rheumatoid arthritis 26'27 and tumour metastasis 28.

Consequences of non-physiological N-glycosylation Glycoproteins d e r i v e d from recombinant DNA will, in many casbs be analogues of the natural molecules. When used as pharmaceutical products, the problems encountered in their administration will be those of any analogue, namely, that the product has a different spectrum of bioactivity, different in-vivo distribution, different circulatory lifetime and different immunogenicity from the native form and, perhaps, new properties. In-vivo administration of recombinant glycoproteins is still in its infancy. At this stage, the consequences of in-vivo administration of a recombinant glycoprotein with non-physiological N-glycosylation can only be inferred. The literature on the properties of oligosaccharides indicates that all the differences in behaviour between the native and recombinant forms of a glycoprotein just mentioned could arise from the presence or absence of certain Nlinked oligosaccharides on the recombinant glycoprotein. Numerous mammalian cell surface lectins 2u have n o w been reported and the distribution of a glycoprotein throughout the body, and its circulatory lifetime, are influenced by the Nlinked oligosaccharides. If the recombinant form carries residues such as outer-arm non-reducing terminal Gal(od-3)Gal or GlcNAc (~1), immune rejection by naturally occurring antibodies, and possible anaphylactic shock, is an immediate possibility. Up to 1% of all circulating serum antibodies in humans are directed against the Gat(od-3)Gal determinant 11. IgE antibodies directed against terminal GlcNAc are present in sera from individuals with bee

venom allergies 3° (the allergenic glycoproteins in bee venom carry Nlinked oligosaccharides with terminal GlcNAc), and immunization with group A streptococcus, a very common cause of infection, elicits IgG antibodies directed against G]cNAc (Ref. 12). Responses to terminal GlcNAc could be a particular problem in monoclonal antibody administration, since IgG naturally carries a small amount of this oligosaccharide determinant. Thus monoclonal antibodies with unusually high levels of GlcNAc could be rejected by naturally occurring anti-GlcNAc antibodies and might also cause pathological symptoms associated with such IgG (Ref. 26). Certain oligosaccharide epitopes are highly immunogenic in mammalian species, for example oligosaccharides carrying a D-xylose residue linked ~1-2 to the ~mannosyl residue and an L-fucose residue linked 0:1-3 to the reducing terminal N-acetylglucosamine residue 31. Yet these two determinants are very commonly found on plant glycoproteins 4, implying that the therapeutic use of mammalian glycoproteins produced in plants may be associated with adverse immunological reactions. There is also increasing evidence that oligosaccharides can affect the antigenic properties of their attached polypeptide 14'32. This implies that a non-physiological change in oligosaccharide may affect the immune response against a native polypeptide, raising the possibility not only of rejection of the administered glycoprotein but also of an autoimmune response against existing native polypeptide. If the recombinant glycoprotein lacks oligosaccharides present on the native form, not only may its distribution and immunogenicity in vivo be different, but also it may not show the f u l l spectrum of biological activities of the native form. Finally, changes in the relative incidence of a constant set of polypeptide-associated Nlinked oligosaccharides can have a profound effect on the biological properties of that glycoprotein 14'26. It cannot, therefore, be assumed that, just because a recombinant glycoprotein carries the same set of

oligosaccharides as the native form (but in altered relative amounts), its biological properties will be the same. Many of these problems may become especially important during long-term repeated or sustained administration, such as replacement therapy, although preliminary results with a recombinant erythropoietin are encouraging 33. These difficulties, essentially arising from our inability to control experimentally the site-specific Nglycosylation of a polypeptide, do not negate efforts to produce recombinant glycoproteins for i n - r i v e administration. Rather they are raised here to highlight s o m e of the potential consequences of administration of a recombinant glycoprotein that may not be revealed during a short-term trial on relatively few individuals, and to stimulate discussion of strategies that may be developed to circumvent some of the potentially more dramatic sideeffects of non-physiological N-glycosylation.

Rationalizing glycosylation In view of the high degree of species-specificity of N-glycosylation, the success or failure of toxicology studies on a recombinant glycoprotein in non-human species cannot be used to predict the consequences of its administration to a human being. Until there are assay systems that accurately predict the consequences of administration of a recombinant glycoprotein to humans, it will be essential to compare the overall N-glycosylation of the recombinant form of a glycoprotein to its appropriate native form. Where the latter is not available, the individual oligosaccharide structures associated with the recombinant glycoprotein should be determined, and the physiological consequences of administering such structures assessed by reference to the wider literature on oligosaccharide function. By identifying the oligosaccharide Structures, strategies can be developed to remodel any potentially harmful determinants [for example enzymic or lectin-assisted removal of Gal(od-3)Gal or Gal(~I-3)GlcNAc

TIBTECH

-

MAY 1989 [Vol. 7]

determinants], or other cell-expression systems chosen. If N-glycosylation is a n a l y s e d sufficiently early in p r o d u c t d e v e l o p m e n t , a rational choice b e t w e e n different expression systems ( w h e t h e r transgenic or cell lines] can be m a d e and the feasibility of a n y n e c e s s a r y r e m o d e l l i n g can be assessed. This w o u l d m i n i m i z e adverse physiological c o n s e q u e n c e s of a d m i n i s t r a t i o n and therefore minimize risk, cost and possible liability at a later stage. Alternative strategies such as genetic m a n i p u l a t i o n leading to e l i m i n a t i o n or r e a r r a n g e m e n t of Nglycosylation sites clearly create non-native, potentially i m m u n o g e n i c polypeptides.

circulatory lifetime. By generating glycoforms, an organism expresses a w h o l e range of natural variants of a p o l y p e p t i d e . P r o d u c t i o n of a m o r e efficacious, n o n - h a r m f u l therapeutic glycoprotein may, in some cases, involve no more than the isolation of a particular natural variant. While the p h e n o m e n o n of Nglycosylation s h o u l d in no w a y interrupt the p r o g r a m m e for developm e n t of r e c o m b i n a n t glycoproteins, it does m e a n that until cell lines are d e v e l o p e d to ensure directed Nglycosylation, it is scientifically and ethically i m p e r a t i v e to define the Nlinked oligosaccharides of a recombinant g l y c o p r o t e i n prior to its in-vivo administration.

Conclusions and future considerations N-linked oligosaccharides, far from being inert and irrelevant c o m p o n e n t s of a glycoprotein, are i m p o r t a n t in defining its structural and biological properties. Certain oligosaccharides are potentially imm u n o g e n i c , others i m m u n o s u p p r e s sive 34'35, and y e t others able to d e t e r m i n e the in-vivo distribution and therefore target destinations of their associated polypeptides. In m a n y cases the bioactivity of a g l y c o p r o t e i n reflects intra- and inter-molecular interactions involving b o t h oligosaccharide and polypeptide. Oligosaccharides are certainly p r o d u c e d in a species-specific and cell-specific way. A r e c o m b i n a n t glycoprotein p r o d u c e d in transgenic systems or cell lines other t h a n those in w h i c h it is n o r m a l l y expressed will have, in m a n y cases, nonphysiological N-glycosylation. For the b i o t e c h n o l o g y and biop h a r m a c e u t i c a l industries, N-glycosytation is a double-edged affair. Ignoring the N-linked oligosaccharides of a r e c o m b i n a n t glycoprotein can lead to an inferior and potentially dangerous product. On the other h a n d a k n o w l e d g e of the structures present can suggest strategies to negate harmful c o n s e q u e n c e s of a d m i n i s t r a t i o n and even to m o d u l a t e the properties of the r e c o m b i n a n t g l y c o p r o t e i n to i m p r o v e its clinical efficacy through, for example, e n h a n c e d bioactivity, specific and controlled targeting, and increased

Acknowledgements RAD, CJE and TWR are members of the Oxford Glycobiology Unit, w h i c h is s u p p o r t e d by Monsanto.

References 1 Montreui], J. (1982) in Comprehensive Biochemistry (Vol. 19B Pt II) (Florkin, M. and Stotz, E. H., eds), pp. 1-188, Elsevier 2 Wagh, P. V. and Bahl, O. P. (1981) CRC Crit. Rev. Biochem. 10,307-377 3 Kobata, A. (1984) in Biology of Carbohydrates (Vol. 2) (Ginsburg, V. and Robbins, P. W., eds), pp. 87-163, Wiley 4 Ashford, D.,-Dwek, R. A., Welply, J. K. eta]. (1987) Eur. J. Biochem. 166, 311-320 5 Homans, S. W., Dwek, R. A. and Rademacher, T. W. (1987) Biochemistry 26, 6571-6578 6 Sutton, B. J. and Phillips, D. C. (1982) Biochem. Soc. Trans. 11,130-132 7 Parekh, R. B., Tse, A. G. D., Dwek, R. A., Williams, A. F. and Rademacherl T. W. (1987) EMBO J. 6, 1233-1244 8 Malaise, M. G., Franchimont, P., Gomez, F., Bouillenne, C. and Mahieu, P. R. (1987) Clin. Immunol. Immunopathol. 45, 1-16 9 Hansen, L., Blue, Y., Barone, K., Collen, D. and Larsen, G. R. (1988) J. Biol. Chem. 263, 15713-15719 10 Schachter, H. (1986) Biochem. Cell. Biol. 64, 163-181 11 Galili, U., Clark, M. R., Shohet, S. B., Buehler, J. and Macher, B. A. (1987) Proc. Natl Acad. Sci. USA 84, 1369-1373

12 Rook, G. A. W., Steele, J. and Rademacher, T. W. (1988) Ann. Rheum. Dis. 47,247-252 13 Swiedler, S. J., Freed, J. H., Tarentino, A. L., Plummer, T. H., Jr and Hart, G. W. (1985) J. Biol. Chem. 260, 4O46-4O54 14 Rademacher, T. W., Parekh, R. B. and Dwek, R. A. (1988) Annu. Rev. Biochem. 57, 785-838 15 Kagawa, Y., Takasaki, S., Utsumi, J. et al. (1988) J. Biol. Chem. 263, 17508-17515 16 Tsuda, E., Goto, M., Murkami, A. et al. (1988) Biochemistry 27, 5646-5654 17 Takeuchi, M., Takasaki, S., Miyazaki, H. et al..(1988) J. Biol. Chem. 263, 3657-3663 18 Gesundheit, N., Fink, D. L., Silverman, L.A. and Weintraub, B. D. (1987) J. Biol. Chem. 262, 5197-5203 19 Olden, K., Parent, J. B. and White, S.L. (1982) Biochim. Biophys. Acta 650, 209-232 20 Ashwell, G. and Harford, J. (1982) Annu. Rev. Biochem. 51, 531-554 21 Chen, H-C., Shimohigashi, Y., Dufen, M.L. and Catt, K.J. (1982) J. Biol. Chem. 257, 14446-14452 22 Matzuk, M. M. and Boime, I. (1988) J. Biol. Chem. 263, 17106-17111 23 Hujj, T. F., Jardieu, P. and Ishizaka, K. (1986)/. Immunol. 131, 1090-1095 24 Sweetnam, P. M. and Tallman, J. F. (1986) Mol. Pharmacol. 29, 299-306 25 Podskalny, J. M., Rouiller, D. G., Grunberger, G. et al. (1986) J. Biol. Chem. 261, 14076-14081 26 Rademacher, T. W., Parekh, R. B., Dwek, R.A. et al. (1988) Springer Semin. Immunopatho]. 10, 231-249 27 Parekh, R. B., Isenberg, D. A., Ansell, B. M. et al. (1988) Lancet i, 966-969 28 Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L. and Kerbel, R. S. (1987) Science 236, 582-585 29 Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560 30 Marz, L., Weber, A., Thalberg, K. and Schroder, H. (1987) Proceedings of the 9th International Symposium on Glycoconjugates (Montreuit, J.,

Verbert, A., Spik, A. and Fournet, B., eds), F32 31 Kaladas, P. M., Goldberg, R. and Poretz, R.D. (1983) Mo]. lmmunol. 20, 727-735 32 Alexander, S. and Eider, J. H. (1984) Science 226, 1328-1330 33 Eschbach, ]. W., Egrie, J. C., Downing, M.R., Browne, J.K. and Adamson, J.W. (1987) N. Engl. J. ivied. 316, 73-78

34 Muchmore, A. V. (1987) J. Immuno]. 35 Muchmore, A. V. (1987) J. Immunol.

and Decker, J. M. 138, 2541 2546 and Decker, J. M. 138, 2547-2553

T I B T E C H - M A Y 1 9 8 9 [Vol. 7]

36 Ferguson, M. A. J., Homans, S. W., Dwek, R. A. and Rademacher, T. W. (1988) Science 239, 753-759 []

[]

[]

37 Saul, F. A., Amzel, L. M. and Poljak, R.J. (1978) J. Biol. Chem. 253, 585-593 []

[]

[]

[]

[]

[]

Genetic engineering for plant oils: potential and limitations James F. Battey, Katherine M. Schmid and John B. Ohlrogge There is considerable interest in the specialty oils markets in modifying oilseed composition to obtain high levels of specific fatty acids. Several oilseed crops are amenable to modern techniques of gone transfer; this suggests that fatty acids now available in small quantities from undomesticated species could be produced by traditional oilseed crops. However, to do this, genes for enzymes of a poorly understood biosynthetic pathway will need to be harnessed. In this review, we discuss progress and problems in the manipulation of plant lipid composition. The oils produced by plants represent a vast renewable resource. World production of plant oils is approximately 60 x 106t with a value of more than $20 x 1 0 9 (Ref. 1). In the USA, about two-thirds of all plant oil is consumed by humans or animals. The remainder serves a wide variety of functions in the industrial sector. The fatty acid composition of an oil determines its physical and chemical properties and thus its uses. The major edible oils have rather similar fatty acid compositions, in which 18 carbon fatty acids with one to three double bonds predominate. Thus, for many edible uses the various major oil sources are interchangeable and their consumption and competitive status in the marketplace is largely controlled by price. However, the specific fatty acid composition of an edible plant oil may sometimes give it special status. In perhaps the most striking instance, the fatty acid profile of cocoa butter confers physical properties desirable

in confectionery manufacture and a price commensurately higher than that of all other plant oils. Furthermore there are grades of cocoa butter: differing fatty acid compositions confer different melting points and this affects the market value ~. Concerns about the possible contributions of saturated fats to heart disease have also put a slight premium on those vegetable oils with the lowest levels of these constituents. For example, the entry of canola oil (low erucic acid rapeseed) into the edible oils market, although dependent on economic factors, has been buoyed by publicity concerning its low (6%) saturated fatty acid content 3.

Desirable products

Most plants, including the major edible oil-producing species, synthesize a limited range of fatty acids: six fatty acids contribute more than 95% of world oil production 4 . Nevertheless, an amazing diversity of structures occurs in the plant kingdom. Surveys of tens of thousands of plant species have revealed nearly a • thousand different fatty acids 5. Some J. F. Battey, K. M. Schmid and J. B. Ohlrogge are at the Department of Botany of these are currently in use by the and Plant Patholo~z, Michigan State chemical industry (Table1), and many more have potential uses but Univezsity, East Lansing, M148824, USA. 1~ 1989, Elsevier Science Publishers Ltd (UK) 0167 - 9430/89/$02,00

38 Weiner, S. J., Kollman, P. A., Case, D. A. et el. (1984) J. Am. Chem. Soc. 106, 765-784 []

[]

[]

are not available in large enough quantities. Progress in the genetic engineering of plants has led to speculation that certain oilseed components could become marketable commodities if the genomes of traditional oilseed crops were modified appropriately. Other goals for this technology include developing alternatives to current imported sources of lauric acid for detergents, or ricinoleic acid for nylons, as well as improved cultivars enriched in individual fatty acids. The viability of current gene transfer technology for oilseeds has been demonstrated by the transformation of rapeseed, flax, cotton, sunflower and safflower ( C a r t h a m u s tinctorius) with foreign DNA (Ref. 6). Seed-specific promoters have been used to ensure that expression of the introduced gene occurs only in the seed 6, increasing the probability that modifications of seed oil may be undertaken without disruption of whole plant physiology. However, the synthesis of oil by plants requires the interaction of multiple gene products. To what degree must the oil synthetic apparatus be modified to bring about industrially applicable changes?

Oil biosynthesis Plants store oil in the form of triacylglycerols (TAG), molecules that have three fatty acids esterified through the three hydroxyl residues of glycerol. The machinery for synthesis of the fatty acids and their incorporation into TAG is distributed between several compartments in plant cells (Fig. 1). The backbones of fatty acid species are produced in the plastids, where a collection of at least six enzymes, termed the fatty acyl synthase, assembles two-carbon units into chains up to 18 carbons long (Fig. 1, reaction a) 7. The primary products of this system in most plants are palmitate and stearate, the 16- and 18-carbon saturated fatty