Eckart Grabenhorst

GENETIC ENGINEERING OF RECOMBINANT GLYCOPROTEINS AND THE GLYCOSYLATION PATHWAY IN MAMMALIAN HOST CELLS

The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation.

The Cytoplasmic, Transmembrane, and Stem Regions of Glycosyltransferases Specify Their in Vivo Functional Sublocalization and Stability in the Golgi

We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human α1,3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human β-trace protein. The sialyl Lewis X:Lewis X ratios detected in β-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human α1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward β-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivofunctional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover.

In Vivo Specificity of Human α1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN β-TRACE PROTEIN

Each of the five human α1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human β-trace protein (β-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc ontoN-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19–52% of the diantennary β-TP N-glycans, and all enzymes were capable of synthesizing the sialyl LewisX (sLex) motif. However, each enzyme produced its own characteristic ratio of sLex/Lex antennae as follows: FT7 (only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto β-TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennaryN-glycans and mono-Lex with monosialo chains. In contrast, FT6 forms mostly α1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.

Stable Expression of the Golgi Form and Secretory Variants of Human Fucosyltransferase III from BHK-21 Cells PURIFICATION AND CHARACTERIZATION OF AN ENGINEERED TRUNCATED FORM FROM THE CULTURE MEDIUM

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human α1,3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galβ1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galβ1-3GlcNAc. The type II motif Galβ1-4GlcNAc in bi-, tri-, or tetraantennary neutral or α2-3/α2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.

Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)-branch of diantennary oligosaccharides from secreted recombinant beta-trace protein.

The human beta-trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of beta-trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2,3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.

Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic.

The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase.

In vitro α1-3 or α1-4 fucosylation of type I and II oligosaccharides with secreted forms of recombinant human fucosyltransferases III and VI

Transgalactosylation of chitobiose and chitotriose employing β-galactosidase from bovine testes yielded mixtures with β1-3 linked galactose (type I) and β1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human α1, 3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewisa structure, whereas approximately 10% fucosylation of the type II isomers to the Lewisx oligosaccharides was observed in long-term incubations.Employing large amounts of human α1, 3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewisx product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely α1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a α1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an α1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewisa oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.

HIGH DENSITY INSECT CELL CULTURE FOR THE PRODUCTION OF RECOMBINANT PROTEINS WITH THE BACULOVIRUS EXPRESSION SYSTEM

Abstract IPLB-Sf21-AE cells (Spodoptera frugiperda) were propagated in stirred tank perfusion bioreactors. A maximum cell density of 5.5×107 viable cells ml−1 was achieved during continuous cultivation in the perfusion mode with medium exchange rates of up to 4 reactor volumes per day. Usually, cells were infected after reaching a concentration of 1 to 3×107 ml−1. The secreted product was harvested continuously, together with spent medium free of cells and cell debris. In addition, more than 99.9 % of the baculovirus was retained within the bioreactor and could be used for subsequent infection of cells. Therefore, cells can be infected with an extremely low multiplicity of infection (MOI) which was usually kept at 0.02. Maximum volumetric production of the recombinant proteins (e.g. human IL-2 glycoforms) was usually achieved 5 to 6 days post infection, one to two days before cell viability dropped drastically because of the virus induced cell lysis. Compared to batch cultures there was only a slight increase of the productivity. However, product was rapidly removed from the bioreactor, thus reducing product degradations by enzymes of lysed cells. In addition, it has been shown that the product was not uniform during the infection phase. In contrast to batch cultures, several harvests containing different microheterogeneous forms of the recombinant protein could be separated.

Expression of human α2, 6-Sialyltransferase in BHK-21A cells increases the sialylation of coexpressed human erythropoietin: NeuAc-transfer onto GalNAc(βl-4)GlcNAc-R motives

We have characterized two BHK cell lines (BHK-21B and BHK-21A) with different glycosylation properties. N-glycans synthesized by BHK-21B cells contain the typical N-acetyllactosamine motif (Gal(βl-4)GlcNAc-R) whereas BHK-21 A cells bear to a high amount nonsialylated terminal GalNAc(βl-4)GlcNAc-R moieties [1]. Due to the incapability of the endogenous oc2, 3-sialyltransferase to transfer NeuAc to the GalNAc(βl-4) GlcNAc-R structure recombinant glycoproteins produced by BHK-21 A cells are under-sialylated.

Construction of novel BHK-21 cell lines coexpressing Golgi resident or soluble forms of human α2,6-sialyltransferase and α1,3/4-fucosyltransferases together with secretory glycoproteins

The most frequently used mammalian host cell lines (BHK-21, CHO) for the production of recombinant therapeutic glycoproteins are incapable of producing α2,6-sialylated or sialyl Lewis X containing Oligosaccharide structures. Therefore, we have constructed novel stable BHK-21 cell lines by cotransfection of cells with plasmids encoding human secretory glycoproteins (β-trace protein, erythropoietin, antithrombin III) and the membrane-bound or soluble forms of human glycosyltransferases α2,6-sialyltransferase (ST6Gal), α1,3-fucosyltransferase III (FT3) or VI (FT6). The secreted recombinant glycoproteins were purified from culture supernatants and were analyzed in detail using HPAE-PAD, mass spectrometry and NMR-analysis of their Oligosaccharide chains. The soluble forms of the recombinant glycosyltransferases showed in vitro activity with Oligosaccharides, glycolipids as well as glycoproteins. However, no modification of coexpressed secretory glycoproteins was detected. Thus a proper targeting/localization of the glycosyltransferases into the appropriate Golgi compartments is required for their in vivo activity on secreted recombinant glycoproteins. Such cell lines can be successfully used for the production of recombinant proteins with novel/tailored glycosylation characteristics that might alter and improve their biological in vivo properties (e.g. stability, tissue addressing).