Mammalian cell gene expression: protein glycosylation Rajesh B. Parekh O x f o r d GlycoSystems Ltd, A b i n g d o n , UK Considerable advances have been made in identifying the factors determining the glycosylation pattern of glycoproteins secreted by mammalian cells. This has allowed a greater appreciation of the way in which recombinant proteins may be glycosylated after expression in a heterologous system. The studies reviewed herein extend the wider view that glycosylation of native and recombinant proteins is a complex event dependent on the protein moiety, the host cell, and also the environment in which transfected cells are cultured. The details of the way in which these factors combine to establish the glycosylation pattern of a secreted protein are now beginning to be unravelled. Current Opinion in Biotechnology 1991, 2:730-734
Introduction The ability to express genes encoding mammalian proteins in heterologous systems has several uses. First, such expression can allow recovery in relatively large amounts of a protein which is naturally scarce, making structural and functional analyses possible. Second, by controlled modification of the coding nucleotide sequence, defined structural variants of the protein can be obtained, and this greatly assists correlation of structure to function. Third, heterologous expression allows proteins of therapeutic value to be obtained in the very large amounts necessary for their use as pharmaceuticals or vaccines. Many of the proteins encoded by mammalian genes, particularly those expressed either at the cell-surface or secreted, are, in their native state, subject to a variety of post-translational modifications, of which the most widespread is glycosylation [ 1 ]. Prokaryotic cells are not capable of attaching either N- or O-glycans to polypeptides in the manner of eukaryotic cells, and the repertoire of glycan structures expressed by the lower eukaryotes (e.g. yeast cells, insect cells, and plant cells) is very limited compared with mammalian cells. Given the importance of protein glycosylation to glycoprotein activity [2], mammalian genes are generally expressed in mammalian cells, and for the reasons given above, such expression is now an integral part of the effort to develop protein-based therapeutics through the application of biotechnology. In this review, recent reports relating to two aspects of protein glycosylation will be discussed. First, the characteristics of protein glycosylation as they are now understood will be summarized.
Second, recent advances in our understanding of the factors influencing the glycosylation pattern of an expressed protein will be reviewed.
Characteristics of protein glycosylation In all mammalian glycoproteins studied to date, several glycan structures are associated with each N-glycosylation site and usually also with each O-glycosylation site. Further, the majority of glycoproteins have several N- and O-glycosylation sites. Even though all these sites are not always fully occupied, glycoproteins generally carry two or more N- and/or O-linked glycans. As a consequence, mammalian glycoproteins occur not as a single discrete structural entity, but rather as a set of glycoforms [2]. Glycoforms of a glycoprotein are defined as structural variants sharing a common amino acid sequence (although not necessarily protein conformation) but differing with respect to the number, structures or location of attached glycans. Furthermore, glycoforms are expressed in a cell type-specific way. For example, Thy-1 is glycosylated very differently when expressed by rat thymocytes and when it is expressed in rat brain, even though the amino acid sequence of both forms is the same [3]. Similarly, numerous other glycoproteins recovered after expression in various mammalian cells are differently glycosylated (e.g. tissue plasminogen activator (t-PA), interferon (IFN)-y, and erythropoietin). Current understanding of the characteristics of protein glycosylation that applies equally to native and recombinant glycoproteins expressed in mammalian cells can be summarized as follows [4..].
Abbreviations CHO~Chinese hamster ovary; G-CSF--granulocyte colony-stimulating factor; GM-CSF~granulocyte-monocytecolony stimulating factor; GnT--N-acetylglucosaminyltransferase;IFN--interferon; II.--interleukin; Neu5Ac--N-acetylneuraminic acid; Neu5GC--N-glycoylneuraminicacid; t-PA---tissue plasminogenactivator.
730
(~) Current Biology Ltd ISSN 0958-1669
Mammalian cell gene expression: protein glycosylation Parekh (a)
Individual glycosylation sites of a protein are associated with several different glycan structures.
(b)
Each glycoprotein usually has several occupied glycosylation sites.
(c)
The occupancy of any individual glycosylation site may be partial (i.e. some glycoprotein molecules carry a glycan at that site, while others do not).
(d)
Cell types differ significantly in their glycosylation capacity: the glycosylation pattern of a glycoprotein obtained after expressing a gene in one cell type will differ from that obtained after expression of the same gene in another cell type.
(e)
The polypeptide structure exerts an effect on the pattem of glycosTlation, but the stringency of this effect is site-specific. For example, certain N-glycosylation sites on a polypeptide are always associated with the same set of glycan structures, irrespective of the cell type in which they are expressed (Asnll7 of t-PA; Asn23 of Thy-1), whereas the glycosytation pattern at other sites on the same polypeptide appears to be totally dependent upon the cell expression system (Asn448 of t-PA; Asn74 and Asn98 of Thy-1).
(f)
Different proteins expressed by the same cell can have quite different glycosylation patterns.
(g)
When cells are maintained under strict homeostasis, the site-specific glycosylation pattern is reproducibly conserved over time and with cellular division.
Factors influencing glycosylation Three factors have now been clearly identified which exert a major influence on the glycosylation of a polypeptide with respect to both the extent of site occupancy and the distribution of glycans at occupied sites. These are (a) the polypeptide structure itself, (b) intracellular, intrinsic parameters (e.g. encoded glycosyl transferase specificities) which are normally invariant and are characteristic of the cell type in question, and (c) environmental factors which affect cellular homeostasis, or in some way change cellular phenotype. The glycowlation pattern of the final expressed form of a protein will be the result of a complex interplay between these three factors.
Effect of polypeptide structure on glycosylation Historically, the evidence suggesting that a polypeptide can influence its own glycosylation has come from two lines of study: those in which different polypeptides simultaneously expressed by the same cellular population were shown to be glycosylated differently, and those in which the same polypeptide expressed in different cell
types (known to vary in their glycosylation capabilities) was shown to be glycosylated in an essentially constant manner. Particularly strong evidence for this has been provided by several recent studies [5..,6",7",8,9,10"]. Lee et al. [5"] fused an IgGl-secreting myeloma with a IgG2b-secreting hybridoma, and compared the glycosylation of the IgG 1 and IgG2b secreted by the parent cells with the IgG 1 homodimers and IgGl-IgG2b heterodimers secreted by daughters of the fusion process. All clones from the fusion produced both homodimers (i.e. IgG1 and IgG2b) and IgOl-IgO2b heterodimers. IgG2b produced by the parent hybridoma was glycosylated differently from the IgG1 produced by the parent myeloma, whereas in heterodimers and homodimers secreted by the fusion cells, the y-chains were glycosylated in the same way as in the parent cell line. In short, the IgG heavy chain was glycosylated in the same way whether it was expressed by a parent cell or by one of the fusion cell clones, even though the fusion cell clones effectively express the glycosylation properties of both parents. Clearly, each heavy chain influences its own glycosylation, and this influence is exerted after assembly of the IgG into two heavy and two light chains. In another study, Fujii et al. [6.] compared the galactosylation of various IgG 1 and IgG2 monoclonals obtained from human myeloma patients. Although individual IgG 1 monoclonals showed minor differences in their glycosylation pattem, certain glycosylation features were common to all, principally the relative galactosylation of mannose Mancz-1 -+ 6 and Mancz-1 --+3 arms. These features were not conserved in IgG2 monoclonals, again indicating subclass-specific glycosylation of IgG heavy chains. Davidson and Castellino [7"] compared the glycosylation of human serum plasminogen with recombinant plasminogen obtained after expression of the cDNA in Chinese hamster ovary (CliO) cells or in insect (SF9) cells. In all three cases, the plasminogen carried one N-glycan. Whereas cell type-specific glycosylation differences were noted, the three forms showed remarkable similarities. For example, all three forms carried sialic acid in cz-2--+ 6 linkage, which is not normally found on CHO- or SF9-derived glycoproteins. This can be interpreted as indicating that the plasminogen polypeptide exerts a very powerful control over its glycosylation. The application of molecular genetics to probing the determinants inherent in an amino acid sequence that are responsible for polypeptide-directed, site-specific glycosylation is proving to be extremely powerful. Two recent studies indicate this, one on human t-PA and one on murine granulocyte-monocyte colony stimulating factor (GM-CSF). The N-glycosylation of several forms of human t-PA, including two recombinant ones, has been extensively studied [8,9]. In all forms studied to date, the N-glycans at Asn117 are of the oligomannose type, and those at site Asn184 are of the complex type. Asnll7 is located in the first Kringle domain, Asn184 in the second; both domains are highly homologous and presumably grossly similar in conformation, so why are the N-glycans at the
731
732
Expressionsystems two sites so differently processed? To study this Wilhelm et al. [10,.] generated a series of variants of human tPA by constructing insertions, duplications and deletions of the domains encoded by the cDNA for human t-PA. All variants were expressed in the same cell line (murine C127 cells), and glycosylation analysis was performed on each variant t-PA glycoprotein. The data clearly indicated that oligomannose structures at Asn117 are obtained only if certain polypeptide regions to the amino-terminal side of this site are retained; without these regions complextype glycans are found at Asn117. The essential region in question would appear from a fine analysis of different deletion mutants to include the amino acids 55-62. Further, in hybrid variants where this sequence was retained but separated from the Asn117-containing Kringle domain by insertion of additional domains, N glycans at Asn117 were not of the oligomannose class. Clearly, a specific (and relatively small) stretch of polypeptide on the amino-terminal side ofAsn117, when present in a precise relative orientation, ensures that only oligomannosetype N-glycans will occur at this site. A study of GM-CSF [11"] shows that a controlling polypeptide region need not be on the amino-terminal side of an N-glycosylation site. A deletion mutant differing in primary structure from native GM-CSF in the carboxyterminal 11 amino acids (terminal 7 deleted, 4 mutated) was transfected into COS-1 cells. The mutant GM-CSF showed a reduced (3000-fold) biological activity and was hyperglycosylated through addition of either larger or more numerous N-glycan chains. It may be through their influence on N-glycosylation that these 11 residues appear to be essential for full biological activity. These resuits obviously have enormous implications for studies in which recombinant DNA methodology is used to pro duce variant proteins for correlating amino acid sequence to function. A change in a property of the recombinant variant cannot be assigned directly to a change in amino acid sequence unless the glycosylation pattern of that particular variant is compared with the glycosylation pattern of other variant forms expressed in the same cell line.
Effect of cell type on protein glycosylation It is now well established that different cell types, whether of the same species or not, can differ significantly with respect to their ability to N-glycosylate proteins [4"',8,9]. These differences can arise from expression by different cell types of different glycosyltransferases and glycosidases. Consequently, the same polypeptide produced in different cell types will usually carry different Y-glycans. The cell-type-specificity of O-glycosylation has not been so fully investigated. Two recent studies [12",13], however, indicate that O-glycosylation features may be much less cell-type-specific. Human granulocyte colony-stimulating factor (G-CSF) [12.] and interleukin (IL)-2 [13] were both expressed in CHO cells. Both polypeptides are in their native state O- (but not N-) glycosylated. In both cases the number, location and apparent structure of attached O-glycans were indistinguishable in the native and recombinant forms.
Two reports [14.,15..] have recently indicated the occurrence of a direct linkage of t-fucose to protein in CHO cells. Both human pro-urokinase [14-] and t-PA [15"'] expressed in CHO cells have been reported to contain Lfucose linked directly to protein through an O-glycosidic linkage. The range of cell types expressing this novel linkage remains to be established, as does its physiological function. Sialylation of the N- and O-glycans of circulating glycoproteins is common, and it is generally considered that such sialylation of recombinant therapeutic glycoproreins increases their circulatory life-time. Sialic acids can occur in a variety of forms, of which N-acetylneuraminic acid (NeuSAc) is the dominant form in humans. Neu5Gc (N-glycoylneuraminic acid) is considered to be an oncofoetal antigen in humans, and in Hanganotziu-Deicher disease (serum-sickness) antibodies are directed against Neu5Gc0~2 ~ 3 Gall31 + 4. It is interesting to note that detection of this determinant by Hokke et al. [16.], in a series of recombinant glycoproteins expressed in CHO cells, identifies this as a determinant for which all CHO-derived recombinant therapeutic glycoproteins should be screened.
Environmental factors affecting protein glycosylation It has emerged clearly during the last few years that the glycosylation pattern of a secreted protein is very sensitive to the physiological status of a cell and to the presence and levels of various extracellular factors [17"]. It has even been argued that monitoring the glycosylation pattern of a secreted protein may serve as a very useful measure of cellular homeostasis. The range of environmental changes that influence cellular glycosylation has been extensively reviewed by Goochee and Monica [17"° ]. Additional recent studies bearing on this issue include the following. Cells in culture are often maintained in medium containing a variety of growth factors and cytokines. In a study [18,,] of the myeloma cell line (OPM-1), it was established that exogenous IL-6 in the medium induced marked and complex changes in the activities of intracellular N-acetylglucosaminyltransferases (GnTs), and that these changes led to alterations in the oligosaccharide structures at the cell surface and presumably also, therefore, of secreted glycoproteins. Specifically, IL-6 reduced GnTIII activity and increased GnT1V and V activities, leading to a shift in expressed complex-type oligosaccharides from bi- to tri- and tetra-antennary structures. The process of transfection can itself induce the expression of previously cryptic glycosyltransferases. In an effort to clone and express a human a-fucosyltransferase from HL-60 cells in CHO cells, Potvin et al. [19"] observed activation of a CliO a-fucosyltransferase, which was previously cryptic, at a frequency comparable to that expected for the transfection of the HL-60 enzyme. The endogenous enzyme was also activated by transfection of CHO DNA into the CHO cell population, confirming that the transfection process p e r se had activated the cryptic enzyme. If this sort of activation is not confined to cz-fucosyltransferases or to CHO cells, and there is no reason
Mammalian cell gene expression: protein glycosylation Parekh 733 to assume that it is, quite widespread changes in cellular glycosylation properties may be induced by the transfection process. It is clearly essential to define the basis of this transferase induction in greater detail and to establish whether any such changes are induced reproducibly. Glycosylation characteristics of a cellular population can also change as a function of age of the culture: Curling et al. [20"] monitored the relative content of glycosylation variants of recombinant human IFN-7 secreted by a CliO cell line grown in serum-free batch culture under controlled conditions. The proportion of non-glycosylated IFN-7 increased continuously by approximately 10 fold from 5% after 3 hours of culture to 30% after 195 hours. The increase in non-glycosylated IFN-7 could not be ascribed to an extracellular enzymatic activity and was independent of the glucose concentration in the medium, but was related to specific cell growth and to IFN-7 production rate.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.
KORNFELD R, KORNFELD S: Assembly of Asparagine-linked Oligosaccharides. A n n u Rev Biocbem 1985, 54:631~544.
2.
RADEMACHERTW, PAREKH RB, DWEK RA: Glycobiology. A n n u Rev Biochem 1988, 57:785--838.
3.
PAREKHRB, TSE AGD, DWEK RA, WILHAMS AF, RADEMACHER TW: Tissue-specific N-glycosylation, Site-specific Glycosylation Patterns and Lentil Recognition of Rat Thy-l. E M B O J 1987, 6:1233-1244.
4. oo
GUMMINGD& Glycosylation of R e c o m b i n a n t Protein Therapeutics: Control and Functional Implications. Glycobiology 1991, 1:115-130. This provides an exhaustive and thorough review of all the aspects of glycosylation that are relevant in the development and production of therapeutic recombinant glycoproteins through biotechnology. Reading this review is strongly recommended. LEE S-O, CONNOLLYJM, RAMIREZ-SOTO D, PORETZ RD: T h e Polypeptide of Immunoglobulin G Influences its Galactosylation I n Vivo. J Biol Chem 1990, 265:5833-5839. The glycosylation patterns of homo- and heterodimeric immunoglobulin G molecules derived from fusion products of an IgGl-producing myeloma and an IgGxb-producing hybridoma were analysed. This is an excellent model system to consider the effects of a polypeptide on its own glycosylation, because variables such as differential rates of synthesis and differences in intracellular compartmentation are eliminated. 5. .,
Conclusions The results summarized herein support the view that the final glycosylation pattem of a protein is the result of the complex interactions between a number of factors. The molecular details of the way in which these factors operate is still poorly defined, but advances in analytical methods and the use of sophisticated molecular genetics suggest that the necessary techniques may now be available to allow a more penetrating analysis of these factors. Two conclusions can nevertheless clearly be drawn. Firstly, that a recombinant glycoprotein will generally be glycosylated in a manner dependent, to a significant extent, on the cell type in which it is expressed (and not in the same way as the native product), and its glycosylation pattern will change if the cellular environment changes during its expression. Secondly, variations in amino acid primary sequence, generated with a view to identifying key amino acids or domains responsible for a particular property, may lead to unexpected changes in glycosylation pattern. It may be these changes in glycosylation rather than in the amino acid sequences p e r se that induce changes in glycoprotein behaviour. The implications of these conclusions for biotechnology studies are considerable. If gene expression in a heterologous system is being used to obtain sufficient quantifies of a glycoprotein for structure-function studies, the recombinant product may not be an accurate model system if it differs significantly from the native form in its glycosytation. Also, the sensitivity of glycosylation to culture environment makes it essential to assess the glycosylation pattern during repeated batch production of a therapeutic glycoprotein. Finally, great care must be taken in ascribing results obtained through the use of protein variants directly to induced changes in amino acid sequence. Such conclusions are tenable only if no changes in glycosylation pattem are associated with the induced changes in amino acid sequence.
6. ,
FUJII S, NISHIURAT, NISHIKAWAA, MIURAR, TAN1GUCH1N: Structural Heterogeneity of Sugar Chains in Immunoglobulin G. J Biol Chem 1990, 265:6009-6018. The glycosylation patterns of different h u m a n monoclonal IgG preparations correlated with the reported specificities on purified oligosaccharides of ~-galactosyl transferases. The data are interpreted to indicate an effect of polypeptide conformation on site-specific N-glycosylation.
DAVIDSON DJ, CASTELLINO FJ: Oligosaccharide Structures Present on Asparagine-289 of Recombinant H u m a n Plasm i n o g e n Expressed in a Chinese Hamster Ovary Cell Line. Biochemistry 1991, 30:625433. This paper is one in a series of incisive studies on the glycosylation of h u m a n plasminogen an excellent model gtycoprotein as it has only one occupied N-glycosylation site. The glycosylation of the recombinant form obtained from CHO ceils was compared with that of the native form and the data indicate both cell-type specific and protein-dependent features of plasminogen glycosylation. 7. o.
8.
PAREKHRB, DWEKRA~ THOMASJR, OPDENAKKERG, RADEMACHER TW, WITI'WERAJ, HOWARD SC, NELSONR, SIEGELNR, JENNINGS MG, ET AL: Ceil Type-specific and Site-specific N-Glycosylation of Type I and Type II H u m a n Tissue Plasminogen Activator. Biochemistry 1989, 28:7644-7662.
9.
PAREKHRB, DWEK RA, RUDD PM, THOMASJR, RADEMACHERWW, WARRENT, WON T-C, HEBERT B, REITZ B, PALMIERM, ETA& NGlycosylation and In Vitro Enzymatic Activity of H u m a n Recombinant Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells and a Murine Cell Line. Bi~ chemistry 1989, 28:7670-7679.
WILHELM J, LEE SG, KALYAN NK, CHENG SM, WEINER F, PIERZCHAIA W, HUNG PP: Alterations in t h e Domain Structure of Tissue-type Plasminogen Activator Change the Nature of Asparagine Glycosylation. Biotechnology 1990, 8:321-325. A s n l l 7 of h u m a n t-PA is normally associated with oligomannose-type glycans irrespective of the cell line from which the t-PA is derived. An analysis of this p h e n o m e n o n using variants generated by recombinant means is reported in this paper. The results suggest a critical role for amino acids distal (amino-terminal side) to ASnll7. 10. ..
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Expression systems LABRANCHECC, CLARKSC, JOHNSON GD, ORNSTEIN D, SABATH DE, TUSHINSKI R, PAETKAU V, PRYSTOWSKY MB: Deletion of Carboxy-terminal Residues of Murine Granulocytemacrophage Colony-stimulating Factor Results in a Loss of Biologic Activity and Altered Glycosylation. Arch Biochem Biophys 1990, 276:153-159. A deletion mutant ofGM CSF differing from native GM-CSF in 11 amino acids at the carboxyl terminus had a profoundly different glycosylation pattern to the native form. Glycosylation can therefore be critically influenced by peptide regions far removed from, and to the carboxy-terminal side of, native GM-CSF. 11. •
12. •
KUBOTAN, ORITA T, HATFORI K, OH-EDA M, OCH1 N, YAMAZAKI T: Structural Characterization of Natural and Recombinant H u m a n Granulocyte Colony-stimulating Factors. J Biochem 1990, 107:486492. Native G CSF carries only O-gtycans. in this paper, O-glycosylation of native and recombinant G-CSF is shown to be very sinailar, suggesting that O-glycosylation may be much less cell type-specific than N-glycosyiation. 13.
YITA N, MAGAZ1NM, MARCHESEE, LUPKERJ, FERRARAP: Closely Related Glycosylation Patterns of Recombinant H u m a n IL-2 Expressed in a CHO Cell Line and Natural IL-2. Lymphokine Res 1990, 9:67-79.
14. •
KENTZEREJ, BUKO A, MENON G, SARINVK: Carbohydrate Composition and Presence of a Fucose-protein Linkage in Recombinant H u m a n Pro-urokinase. Biochem Biophys Res Commun 1990, 171:401-406. A novel linkage between fucose and protein is demonstrated by isolation of a glycopeptide from recombinant h u m a n pro-urokinase (expressed in CHO cells) which contains fucose covalently attached to protein.
from CHO cells. This form of sialic acid is an oncofoetal antigen in humans and is the antigen in certain serum-sickness disorders. 17. GOOCHEECF, MONICA T: Environmental Effects on Protein .. Glycosylation. Biotechnology 1990, 8:421-427. The first exhaustive review of the effects of changes in various extracellular parameters on the gtycosylation pattern of secreted and surfaceassociated glycoproteins. A quite outstanding article, which is now essential reading. 18. oo
NAKAOH, NISHIKAWAA, KARASHUNOT, NISHIURA T, IIDA M, KANAYAMA Y, YONEZAWAT, TARU1S, TANIGUCHIN: Modulation of N-acetylglucosaminyltransferase III, IV and V Activities and Alterations of t h e Surface Oligosaccharide Structures of a Myeloma Cell Line by Interleukin 6. Biochem Biophys Res Commun 1990, 172:1260-1266. It has often been suggested that cytokines influence the glycosylation properties of a cell. This paper provides very strong evidence that exogenously added IL-6 changes the spectrum of intracellular glycosyltransferases activities and that these changes lead to a new distribution of cell surface oligosaccharides. 19. ••
POTVlNB, KUMAR R, HOWARD DR, STANLEY P: Transfection of a H u m a n cz-(1,3) Fucosyltransferase Gene into Chinese Hamster Ovary Cells. J Biol Chem 1990, 265:1615-1622. In an effort to clone and express h u m a n a-1(1,3) fucosyttransferase in CHO cells, several CHO transfectants expressing this enzyme activity but not containing the human enzyme were obtained. Induction of the cryptic CHO enzyme could also be induced by transfecting CHO cells with CHO DNA, suggesting that the process of transfection per se led to activation of a cryptic gene. This has obvious implications for transfection of cells in general.
HARRISRJ, LEONARDCK, GUZZETrA AN, SPELLMANMW: Tissue Plasminogen Activator has an O-litlked Fucose Attached to Threonine 61 in the Epidermal Growth Factor Domain. Biochemistry 1991, 30:2311-2314. The novel covalent linkage through an O-glycosidic bond of Lfucose to the Thr61 side-chain of recombinant h u m a n t-PA (expressed in CHO cells) is establshed by direct carbohydrate analysis and mass spectrometry on tryptic and chymotryptic peptides.
CURLING EMA, I-IAYTER PM, BAINES AJ, BULL AT, GULL K, STRANGEPG, JENKINS N: Recombinant H u m a n Interferon-T. Biochem J 1990, 272:333-337. The relative content of non-glycosylated IFN-'f in the secreted pool of recombinant h u m a n IFN-7, monitored as a function of cell culture age, cell specific growth, and IFN-'f secretion rate was found to increase approximately 10-fold after 195 hours of contip.uous batch culture. This implies that the n u m b e r of N-glycan chains, in addition to the precise structures of these chains, is dependent on the physiological status of a cell population.
HOKKECH, BEROWERFF AA, VAN DEDEM GWK, OOSTRUMJV, KAMERLINGJP, VLIEGEN'I1-1ARDTJFG: Sialylated Carbohydrate Chains of Recombinant H u m a n Glycoproteins Expressed in Chinese Hamster Ovary Cells Contain Traces of N-Glycolylneuraminic Acid. FEBS Lett 1990, 275:9-14. This paper reports the presence (at a level of 3%) of N-glycolylneuraminic acid on four recombinant forms of h u m a n proteins derived
RB Parekh, Oxford Glycosystems Ltd, Unit 4 Hitching Court, BlacMands Way, Abingdon, Oxon, OX14 1RG, UK.
15. .,,
20. •
16.
•
Introduction The ability to express genes encoding mammalian proteins in heterologous systems has several uses. First, such expression can allow recovery in relatively large amounts of a protein which is naturally scarce, making structural and functional analyses possible. Second, by controlled modification of the coding nucleotide sequence, defined structural variants of the protein can be obtained, and this greatly assists correlation of structure to function. Third, heterologous expression allows proteins of therapeutic value to be obtained in the very large amounts necessary for their use as pharmaceuticals or vaccines. Many of the proteins encoded by mammalian genes, particularly those expressed either at the cell-surface or secreted, are, in their native state, subject to a variety of post-translational modifications, of which the most widespread is glycosylation [ 1 ]. Prokaryotic cells are not capable of attaching either N- or O-glycans to polypeptides in the manner of eukaryotic cells, and the repertoire of glycan structures expressed by the lower eukaryotes (e.g. yeast cells, insect cells, and plant cells) is very limited compared with mammalian cells. Given the importance of protein glycosylation to glycoprotein activity [2], mammalian genes are generally expressed in mammalian cells, and for the reasons given above, such expression is now an integral part of the effort to develop protein-based therapeutics through the application of biotechnology. In this review, recent reports relating to two aspects of protein glycosylation will be discussed. First, the characteristics of protein glycosylation as they are now understood will be summarized.
Second, recent advances in our understanding of the factors influencing the glycosylation pattern of an expressed protein will be reviewed.
Characteristics of protein glycosylation In all mammalian glycoproteins studied to date, several glycan structures are associated with each N-glycosylation site and usually also with each O-glycosylation site. Further, the majority of glycoproteins have several N- and O-glycosylation sites. Even though all these sites are not always fully occupied, glycoproteins generally carry two or more N- and/or O-linked glycans. As a consequence, mammalian glycoproteins occur not as a single discrete structural entity, but rather as a set of glycoforms [2]. Glycoforms of a glycoprotein are defined as structural variants sharing a common amino acid sequence (although not necessarily protein conformation) but differing with respect to the number, structures or location of attached glycans. Furthermore, glycoforms are expressed in a cell type-specific way. For example, Thy-1 is glycosylated very differently when expressed by rat thymocytes and when it is expressed in rat brain, even though the amino acid sequence of both forms is the same [3]. Similarly, numerous other glycoproteins recovered after expression in various mammalian cells are differently glycosylated (e.g. tissue plasminogen activator (t-PA), interferon (IFN)-y, and erythropoietin). Current understanding of the characteristics of protein glycosylation that applies equally to native and recombinant glycoproteins expressed in mammalian cells can be summarized as follows [4..].
Abbreviations CHO~Chinese hamster ovary; G-CSF--granulocyte colony-stimulating factor; GM-CSF~granulocyte-monocytecolony stimulating factor; GnT--N-acetylglucosaminyltransferase;IFN--interferon; II.--interleukin; Neu5Ac--N-acetylneuraminic acid; Neu5GC--N-glycoylneuraminicacid; t-PA---tissue plasminogenactivator.
730
(~) Current Biology Ltd ISSN 0958-1669
Mammalian cell gene expression: protein glycosylation Parekh (a)
Individual glycosylation sites of a protein are associated with several different glycan structures.
(b)
Each glycoprotein usually has several occupied glycosylation sites.
(c)
The occupancy of any individual glycosylation site may be partial (i.e. some glycoprotein molecules carry a glycan at that site, while others do not).
(d)
Cell types differ significantly in their glycosylation capacity: the glycosylation pattern of a glycoprotein obtained after expressing a gene in one cell type will differ from that obtained after expression of the same gene in another cell type.
(e)
The polypeptide structure exerts an effect on the pattem of glycosTlation, but the stringency of this effect is site-specific. For example, certain N-glycosylation sites on a polypeptide are always associated with the same set of glycan structures, irrespective of the cell type in which they are expressed (Asnll7 of t-PA; Asn23 of Thy-1), whereas the glycosytation pattern at other sites on the same polypeptide appears to be totally dependent upon the cell expression system (Asn448 of t-PA; Asn74 and Asn98 of Thy-1).
(f)
Different proteins expressed by the same cell can have quite different glycosylation patterns.
(g)
When cells are maintained under strict homeostasis, the site-specific glycosylation pattern is reproducibly conserved over time and with cellular division.
Factors influencing glycosylation Three factors have now been clearly identified which exert a major influence on the glycosylation of a polypeptide with respect to both the extent of site occupancy and the distribution of glycans at occupied sites. These are (a) the polypeptide structure itself, (b) intracellular, intrinsic parameters (e.g. encoded glycosyl transferase specificities) which are normally invariant and are characteristic of the cell type in question, and (c) environmental factors which affect cellular homeostasis, or in some way change cellular phenotype. The glycowlation pattern of the final expressed form of a protein will be the result of a complex interplay between these three factors.
Effect of polypeptide structure on glycosylation Historically, the evidence suggesting that a polypeptide can influence its own glycosylation has come from two lines of study: those in which different polypeptides simultaneously expressed by the same cellular population were shown to be glycosylated differently, and those in which the same polypeptide expressed in different cell
types (known to vary in their glycosylation capabilities) was shown to be glycosylated in an essentially constant manner. Particularly strong evidence for this has been provided by several recent studies [5..,6",7",8,9,10"]. Lee et al. [5"] fused an IgGl-secreting myeloma with a IgG2b-secreting hybridoma, and compared the glycosylation of the IgG 1 and IgG2b secreted by the parent cells with the IgG 1 homodimers and IgGl-IgG2b heterodimers secreted by daughters of the fusion process. All clones from the fusion produced both homodimers (i.e. IgG1 and IgG2b) and IgOl-IgO2b heterodimers. IgG2b produced by the parent hybridoma was glycosylated differently from the IgG1 produced by the parent myeloma, whereas in heterodimers and homodimers secreted by the fusion cells, the y-chains were glycosylated in the same way as in the parent cell line. In short, the IgG heavy chain was glycosylated in the same way whether it was expressed by a parent cell or by one of the fusion cell clones, even though the fusion cell clones effectively express the glycosylation properties of both parents. Clearly, each heavy chain influences its own glycosylation, and this influence is exerted after assembly of the IgG into two heavy and two light chains. In another study, Fujii et al. [6.] compared the galactosylation of various IgG 1 and IgG2 monoclonals obtained from human myeloma patients. Although individual IgG 1 monoclonals showed minor differences in their glycosylation pattem, certain glycosylation features were common to all, principally the relative galactosylation of mannose Mancz-1 -+ 6 and Mancz-1 --+3 arms. These features were not conserved in IgG2 monoclonals, again indicating subclass-specific glycosylation of IgG heavy chains. Davidson and Castellino [7"] compared the glycosylation of human serum plasminogen with recombinant plasminogen obtained after expression of the cDNA in Chinese hamster ovary (CliO) cells or in insect (SF9) cells. In all three cases, the plasminogen carried one N-glycan. Whereas cell type-specific glycosylation differences were noted, the three forms showed remarkable similarities. For example, all three forms carried sialic acid in cz-2--+ 6 linkage, which is not normally found on CHO- or SF9-derived glycoproteins. This can be interpreted as indicating that the plasminogen polypeptide exerts a very powerful control over its glycosylation. The application of molecular genetics to probing the determinants inherent in an amino acid sequence that are responsible for polypeptide-directed, site-specific glycosylation is proving to be extremely powerful. Two recent studies indicate this, one on human t-PA and one on murine granulocyte-monocyte colony stimulating factor (GM-CSF). The N-glycosylation of several forms of human t-PA, including two recombinant ones, has been extensively studied [8,9]. In all forms studied to date, the N-glycans at Asn117 are of the oligomannose type, and those at site Asn184 are of the complex type. Asnll7 is located in the first Kringle domain, Asn184 in the second; both domains are highly homologous and presumably grossly similar in conformation, so why are the N-glycans at the
731
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Expressionsystems two sites so differently processed? To study this Wilhelm et al. [10,.] generated a series of variants of human tPA by constructing insertions, duplications and deletions of the domains encoded by the cDNA for human t-PA. All variants were expressed in the same cell line (murine C127 cells), and glycosylation analysis was performed on each variant t-PA glycoprotein. The data clearly indicated that oligomannose structures at Asn117 are obtained only if certain polypeptide regions to the amino-terminal side of this site are retained; without these regions complextype glycans are found at Asn117. The essential region in question would appear from a fine analysis of different deletion mutants to include the amino acids 55-62. Further, in hybrid variants where this sequence was retained but separated from the Asn117-containing Kringle domain by insertion of additional domains, N glycans at Asn117 were not of the oligomannose class. Clearly, a specific (and relatively small) stretch of polypeptide on the amino-terminal side ofAsn117, when present in a precise relative orientation, ensures that only oligomannosetype N-glycans will occur at this site. A study of GM-CSF [11"] shows that a controlling polypeptide region need not be on the amino-terminal side of an N-glycosylation site. A deletion mutant differing in primary structure from native GM-CSF in the carboxyterminal 11 amino acids (terminal 7 deleted, 4 mutated) was transfected into COS-1 cells. The mutant GM-CSF showed a reduced (3000-fold) biological activity and was hyperglycosylated through addition of either larger or more numerous N-glycan chains. It may be through their influence on N-glycosylation that these 11 residues appear to be essential for full biological activity. These resuits obviously have enormous implications for studies in which recombinant DNA methodology is used to pro duce variant proteins for correlating amino acid sequence to function. A change in a property of the recombinant variant cannot be assigned directly to a change in amino acid sequence unless the glycosylation pattern of that particular variant is compared with the glycosylation pattern of other variant forms expressed in the same cell line.
Effect of cell type on protein glycosylation It is now well established that different cell types, whether of the same species or not, can differ significantly with respect to their ability to N-glycosylate proteins [4"',8,9]. These differences can arise from expression by different cell types of different glycosyltransferases and glycosidases. Consequently, the same polypeptide produced in different cell types will usually carry different Y-glycans. The cell-type-specificity of O-glycosylation has not been so fully investigated. Two recent studies [12",13], however, indicate that O-glycosylation features may be much less cell-type-specific. Human granulocyte colony-stimulating factor (G-CSF) [12.] and interleukin (IL)-2 [13] were both expressed in CHO cells. Both polypeptides are in their native state O- (but not N-) glycosylated. In both cases the number, location and apparent structure of attached O-glycans were indistinguishable in the native and recombinant forms.
Two reports [14.,15..] have recently indicated the occurrence of a direct linkage of t-fucose to protein in CHO cells. Both human pro-urokinase [14-] and t-PA [15"'] expressed in CHO cells have been reported to contain Lfucose linked directly to protein through an O-glycosidic linkage. The range of cell types expressing this novel linkage remains to be established, as does its physiological function. Sialylation of the N- and O-glycans of circulating glycoproteins is common, and it is generally considered that such sialylation of recombinant therapeutic glycoproreins increases their circulatory life-time. Sialic acids can occur in a variety of forms, of which N-acetylneuraminic acid (NeuSAc) is the dominant form in humans. Neu5Gc (N-glycoylneuraminic acid) is considered to be an oncofoetal antigen in humans, and in Hanganotziu-Deicher disease (serum-sickness) antibodies are directed against Neu5Gc0~2 ~ 3 Gall31 + 4. It is interesting to note that detection of this determinant by Hokke et al. [16.], in a series of recombinant glycoproteins expressed in CHO cells, identifies this as a determinant for which all CHO-derived recombinant therapeutic glycoproteins should be screened.
Environmental factors affecting protein glycosylation It has emerged clearly during the last few years that the glycosylation pattern of a secreted protein is very sensitive to the physiological status of a cell and to the presence and levels of various extracellular factors [17"]. It has even been argued that monitoring the glycosylation pattern of a secreted protein may serve as a very useful measure of cellular homeostasis. The range of environmental changes that influence cellular glycosylation has been extensively reviewed by Goochee and Monica [17"° ]. Additional recent studies bearing on this issue include the following. Cells in culture are often maintained in medium containing a variety of growth factors and cytokines. In a study [18,,] of the myeloma cell line (OPM-1), it was established that exogenous IL-6 in the medium induced marked and complex changes in the activities of intracellular N-acetylglucosaminyltransferases (GnTs), and that these changes led to alterations in the oligosaccharide structures at the cell surface and presumably also, therefore, of secreted glycoproteins. Specifically, IL-6 reduced GnTIII activity and increased GnT1V and V activities, leading to a shift in expressed complex-type oligosaccharides from bi- to tri- and tetra-antennary structures. The process of transfection can itself induce the expression of previously cryptic glycosyltransferases. In an effort to clone and express a human a-fucosyltransferase from HL-60 cells in CHO cells, Potvin et al. [19"] observed activation of a CliO a-fucosyltransferase, which was previously cryptic, at a frequency comparable to that expected for the transfection of the HL-60 enzyme. The endogenous enzyme was also activated by transfection of CHO DNA into the CHO cell population, confirming that the transfection process p e r se had activated the cryptic enzyme. If this sort of activation is not confined to cz-fucosyltransferases or to CHO cells, and there is no reason
Mammalian cell gene expression: protein glycosylation Parekh 733 to assume that it is, quite widespread changes in cellular glycosylation properties may be induced by the transfection process. It is clearly essential to define the basis of this transferase induction in greater detail and to establish whether any such changes are induced reproducibly. Glycosylation characteristics of a cellular population can also change as a function of age of the culture: Curling et al. [20"] monitored the relative content of glycosylation variants of recombinant human IFN-7 secreted by a CliO cell line grown in serum-free batch culture under controlled conditions. The proportion of non-glycosylated IFN-7 increased continuously by approximately 10 fold from 5% after 3 hours of culture to 30% after 195 hours. The increase in non-glycosylated IFN-7 could not be ascribed to an extracellular enzymatic activity and was independent of the glucose concentration in the medium, but was related to specific cell growth and to IFN-7 production rate.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.
KORNFELD R, KORNFELD S: Assembly of Asparagine-linked Oligosaccharides. A n n u Rev Biocbem 1985, 54:631~544.
2.
RADEMACHERTW, PAREKH RB, DWEK RA: Glycobiology. A n n u Rev Biochem 1988, 57:785--838.
3.
PAREKHRB, TSE AGD, DWEK RA, WILHAMS AF, RADEMACHER TW: Tissue-specific N-glycosylation, Site-specific Glycosylation Patterns and Lentil Recognition of Rat Thy-l. E M B O J 1987, 6:1233-1244.
4. oo
GUMMINGD& Glycosylation of R e c o m b i n a n t Protein Therapeutics: Control and Functional Implications. Glycobiology 1991, 1:115-130. This provides an exhaustive and thorough review of all the aspects of glycosylation that are relevant in the development and production of therapeutic recombinant glycoproteins through biotechnology. Reading this review is strongly recommended. LEE S-O, CONNOLLYJM, RAMIREZ-SOTO D, PORETZ RD: T h e Polypeptide of Immunoglobulin G Influences its Galactosylation I n Vivo. J Biol Chem 1990, 265:5833-5839. The glycosylation patterns of homo- and heterodimeric immunoglobulin G molecules derived from fusion products of an IgGl-producing myeloma and an IgGxb-producing hybridoma were analysed. This is an excellent model system to consider the effects of a polypeptide on its own glycosylation, because variables such as differential rates of synthesis and differences in intracellular compartmentation are eliminated. 5. .,
Conclusions The results summarized herein support the view that the final glycosylation pattem of a protein is the result of the complex interactions between a number of factors. The molecular details of the way in which these factors operate is still poorly defined, but advances in analytical methods and the use of sophisticated molecular genetics suggest that the necessary techniques may now be available to allow a more penetrating analysis of these factors. Two conclusions can nevertheless clearly be drawn. Firstly, that a recombinant glycoprotein will generally be glycosylated in a manner dependent, to a significant extent, on the cell type in which it is expressed (and not in the same way as the native product), and its glycosylation pattern will change if the cellular environment changes during its expression. Secondly, variations in amino acid primary sequence, generated with a view to identifying key amino acids or domains responsible for a particular property, may lead to unexpected changes in glycosylation pattern. It may be these changes in glycosylation rather than in the amino acid sequences p e r se that induce changes in glycoprotein behaviour. The implications of these conclusions for biotechnology studies are considerable. If gene expression in a heterologous system is being used to obtain sufficient quantifies of a glycoprotein for structure-function studies, the recombinant product may not be an accurate model system if it differs significantly from the native form in its glycosytation. Also, the sensitivity of glycosylation to culture environment makes it essential to assess the glycosylation pattern during repeated batch production of a therapeutic glycoprotein. Finally, great care must be taken in ascribing results obtained through the use of protein variants directly to induced changes in amino acid sequence. Such conclusions are tenable only if no changes in glycosylation pattem are associated with the induced changes in amino acid sequence.
6. ,
FUJII S, NISHIURAT, NISHIKAWAA, MIURAR, TAN1GUCH1N: Structural Heterogeneity of Sugar Chains in Immunoglobulin G. J Biol Chem 1990, 265:6009-6018. The glycosylation patterns of different h u m a n monoclonal IgG preparations correlated with the reported specificities on purified oligosaccharides of ~-galactosyl transferases. The data are interpreted to indicate an effect of polypeptide conformation on site-specific N-glycosylation.
DAVIDSON DJ, CASTELLINO FJ: Oligosaccharide Structures Present on Asparagine-289 of Recombinant H u m a n Plasm i n o g e n Expressed in a Chinese Hamster Ovary Cell Line. Biochemistry 1991, 30:625433. This paper is one in a series of incisive studies on the glycosylation of h u m a n plasminogen an excellent model gtycoprotein as it has only one occupied N-glycosylation site. The glycosylation of the recombinant form obtained from CHO ceils was compared with that of the native form and the data indicate both cell-type specific and protein-dependent features of plasminogen glycosylation. 7. o.
8.
PAREKHRB, DWEKRA~ THOMASJR, OPDENAKKERG, RADEMACHER TW, WITI'WERAJ, HOWARD SC, NELSONR, SIEGELNR, JENNINGS MG, ET AL: Ceil Type-specific and Site-specific N-Glycosylation of Type I and Type II H u m a n Tissue Plasminogen Activator. Biochemistry 1989, 28:7644-7662.
9.
PAREKHRB, DWEK RA, RUDD PM, THOMASJR, RADEMACHERWW, WARRENT, WON T-C, HEBERT B, REITZ B, PALMIERM, ETA& NGlycosylation and In Vitro Enzymatic Activity of H u m a n Recombinant Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells and a Murine Cell Line. Bi~ chemistry 1989, 28:7670-7679.
WILHELM J, LEE SG, KALYAN NK, CHENG SM, WEINER F, PIERZCHAIA W, HUNG PP: Alterations in t h e Domain Structure of Tissue-type Plasminogen Activator Change the Nature of Asparagine Glycosylation. Biotechnology 1990, 8:321-325. A s n l l 7 of h u m a n t-PA is normally associated with oligomannose-type glycans irrespective of the cell line from which the t-PA is derived. An analysis of this p h e n o m e n o n using variants generated by recombinant means is reported in this paper. The results suggest a critical role for amino acids distal (amino-terminal side) to ASnll7. 10. ..
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Expression systems LABRANCHECC, CLARKSC, JOHNSON GD, ORNSTEIN D, SABATH DE, TUSHINSKI R, PAETKAU V, PRYSTOWSKY MB: Deletion of Carboxy-terminal Residues of Murine Granulocytemacrophage Colony-stimulating Factor Results in a Loss of Biologic Activity and Altered Glycosylation. Arch Biochem Biophys 1990, 276:153-159. A deletion mutant ofGM CSF differing from native GM-CSF in 11 amino acids at the carboxyl terminus had a profoundly different glycosylation pattern to the native form. Glycosylation can therefore be critically influenced by peptide regions far removed from, and to the carboxy-terminal side of, native GM-CSF. 11. •
12. •
KUBOTAN, ORITA T, HATFORI K, OH-EDA M, OCH1 N, YAMAZAKI T: Structural Characterization of Natural and Recombinant H u m a n Granulocyte Colony-stimulating Factors. J Biochem 1990, 107:486492. Native G CSF carries only O-gtycans. in this paper, O-glycosylation of native and recombinant G-CSF is shown to be very sinailar, suggesting that O-glycosylation may be much less cell type-specific than N-glycosyiation. 13.
YITA N, MAGAZ1NM, MARCHESEE, LUPKERJ, FERRARAP: Closely Related Glycosylation Patterns of Recombinant H u m a n IL-2 Expressed in a CHO Cell Line and Natural IL-2. Lymphokine Res 1990, 9:67-79.
14. •
KENTZEREJ, BUKO A, MENON G, SARINVK: Carbohydrate Composition and Presence of a Fucose-protein Linkage in Recombinant H u m a n Pro-urokinase. Biochem Biophys Res Commun 1990, 171:401-406. A novel linkage between fucose and protein is demonstrated by isolation of a glycopeptide from recombinant h u m a n pro-urokinase (expressed in CHO cells) which contains fucose covalently attached to protein.
from CHO cells. This form of sialic acid is an oncofoetal antigen in humans and is the antigen in certain serum-sickness disorders. 17. GOOCHEECF, MONICA T: Environmental Effects on Protein .. Glycosylation. Biotechnology 1990, 8:421-427. The first exhaustive review of the effects of changes in various extracellular parameters on the gtycosylation pattern of secreted and surfaceassociated glycoproteins. A quite outstanding article, which is now essential reading. 18. oo
NAKAOH, NISHIKAWAA, KARASHUNOT, NISHIURA T, IIDA M, KANAYAMA Y, YONEZAWAT, TARU1S, TANIGUCHIN: Modulation of N-acetylglucosaminyltransferase III, IV and V Activities and Alterations of t h e Surface Oligosaccharide Structures of a Myeloma Cell Line by Interleukin 6. Biochem Biophys Res Commun 1990, 172:1260-1266. It has often been suggested that cytokines influence the glycosylation properties of a cell. This paper provides very strong evidence that exogenously added IL-6 changes the spectrum of intracellular glycosyltransferases activities and that these changes lead to a new distribution of cell surface oligosaccharides. 19. ••
POTVlNB, KUMAR R, HOWARD DR, STANLEY P: Transfection of a H u m a n cz-(1,3) Fucosyltransferase Gene into Chinese Hamster Ovary Cells. J Biol Chem 1990, 265:1615-1622. In an effort to clone and express h u m a n a-1(1,3) fucosyttransferase in CHO cells, several CHO transfectants expressing this enzyme activity but not containing the human enzyme were obtained. Induction of the cryptic CHO enzyme could also be induced by transfecting CHO cells with CHO DNA, suggesting that the process of transfection per se led to activation of a cryptic gene. This has obvious implications for transfection of cells in general.
HARRISRJ, LEONARDCK, GUZZETrA AN, SPELLMANMW: Tissue Plasminogen Activator has an O-litlked Fucose Attached to Threonine 61 in the Epidermal Growth Factor Domain. Biochemistry 1991, 30:2311-2314. The novel covalent linkage through an O-glycosidic bond of Lfucose to the Thr61 side-chain of recombinant h u m a n t-PA (expressed in CHO cells) is establshed by direct carbohydrate analysis and mass spectrometry on tryptic and chymotryptic peptides.
CURLING EMA, I-IAYTER PM, BAINES AJ, BULL AT, GULL K, STRANGEPG, JENKINS N: Recombinant H u m a n Interferon-T. Biochem J 1990, 272:333-337. The relative content of non-glycosylated IFN-'f in the secreted pool of recombinant h u m a n IFN-7, monitored as a function of cell culture age, cell specific growth, and IFN-'f secretion rate was found to increase approximately 10-fold after 195 hours of contip.uous batch culture. This implies that the n u m b e r of N-glycan chains, in addition to the precise structures of these chains, is dependent on the physiological status of a cell population.
HOKKECH, BEROWERFF AA, VAN DEDEM GWK, OOSTRUMJV, KAMERLINGJP, VLIEGEN'I1-1ARDTJFG: Sialylated Carbohydrate Chains of Recombinant H u m a n Glycoproteins Expressed in Chinese Hamster Ovary Cells Contain Traces of N-Glycolylneuraminic Acid. FEBS Lett 1990, 275:9-14. This paper reports the presence (at a level of 3%) of N-glycolylneuraminic acid on four recombinant forms of h u m a n proteins derived
RB Parekh, Oxford Glycosystems Ltd, Unit 4 Hitching Court, BlacMands Way, Abingdon, Oxon, OX14 1RG, UK.
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