Recombinant glycoproteins with reduced antennary fucosylation

Abstract

The present invention relates to methods for reducing antennary fucosylation of complex N-glycans in recombinantly expressed glycoproteins, cell lines that can be used in said methods, respective recombinant glycoproteins, and methods for expressing the same in said cell lines.

Claims

1. A method for reducing antennary fucosylation of complex N-glycans in a glycoprotein that is recombinantly expressed in a human primary amniocyte cell line comprising at least one nucleic acid encoding gene products of the adenoviral E1 and pIX regions, comprising the step of overexpressing together with the glycoprotein a β-galactoside α-2,6-sialyltransferase 1 (ST6Gal1) and a β-galactoside α-2,3-sialyltransferase 4 (ST3Gal4).

2. The method of claim 1, wherein the glycoprotein is characterized by an at least 80% reduction in antennary fucosylation of complex N-glycans as compared to the same recombinant glycoprotein expressed without overexpression of the ST6Gal1 and the ST3Gal4.

3. The method of claim 1, wherein at least 80% of the complex N-glycan antennae of the recombinantly expressed glycoprotein are not fucosylated.

4. The method of claim 1, wherein the glycoprotein is selected from the group consisting of α1-antitrypsin (AAT), hepatocyte growth factor (HGF), Factor VII (FVII), Factor VIII (FVIII), Factor IX (FIX), von Willebrand-Factor (vWF), alkaline phosphatase, and C1 esterase inhibitor (C1-inhibitor; C1 Inh).

5. The method of claim 1, wherein the glycoprotein is a mammalian.

6. The method of claim 5, wherein the glycoprotein is a human glycoprotein.

7. The method of claim 1, further comprising (i) a β-galactoside α-2,3-sialyltransferase 1 (ST3Gal1) wherein the ST3Gal1 is not overexpressed together with the glycoprotein, and/or (ii) the expression of an α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase A (GnTIVa), an α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase B (GnTIVb), and an α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase A (GnTV) are not reduced.

8. A cell line that is genetically modified to overexpress β-galactoside α-2,6-sialyltransferase 1 (ST6Gal1) and α-2,3-sialyltransferase 4 (ST3Gal4), and wherein the cell line is a human primary amniocyte cell line comprising at least one nucleic acid encoding the gene products of the adenoviral E1 and pIX regions.

9. The cell line of claim 8, wherein the cell line comprises endogenous gene(s) encoding the ST6Gal1 and the ST3Gal4, and further has at least one genetic element, selected from the group consisting of a promoter, an enhancing element, and a stabilizing element inserted into the genome in one or more position(s) suitable to cause overexpression of the ST6Gal1 and the ST3Gal4.

10. The cell line of claim 8, wherein the cell line comprises exogenous nucleic acid(s) encoding the ST6Gal1 and the ST3Gal4.

11. The cell line of claim 8, wherein the cell line is derived from a CAP cell line.

12. The cell line of claim 8, wherein the cell line is not genetically modified to (i) overexpress a β-galactoside α-2,3-sialyltransferase 1 (ST3Gal1), and/or (ii) reduce the expression of an α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase A (GnTIVa), an α-1,3-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase B (GnTIVb), and an α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase A (GnTV).

13. A recombinant glycoprotein having complex N-glycans, wherein antennary fucosylation of the complex N-glycans is reduced, so that at least 80% of the complex N-glycan antennae of the recombinant glycoprotein are not fucosylated, wherein said glycoprotein us is produced in a cell line according to claim 8.

14. The recombinant glycoprotein of claim 13, wherein the glycoprotein is selected from the group consisting of α1-antitrypsin (AAT), hepatocyte growth factor (HGF), Factor VII (FVII), Factor VIII (FVIII), Factor IX (FIX), von Willebrand-Factor (vWF), alkaline phosphatase, and C1 esterase inhibitor (C1-inhibitor; C1 Inh).

15. A method for the expression of a recombinant glycoprotein of claim 13, comprising the steps of: (a) providing a cell line that is genetically modified to overexpress a β-galactoside α-2,6-sialyltransferase 1 (ST6Gal1) and a α-2,3-sialyltransferase 4 (ST3Gal4), and wherein the cell line is a human primary amniocyte cell line comprising at least one nucleic acid encoding the gene products of the adenoviral E1 and pIX regions, (b) expressing the recombinant glycoprotein in said cell line, wherein the antennary fucosylation of the N-glycans of the glycoprotein is reduced; and (c) isolating the recombinant glycoprotein from the cells or the cell culture supernatant.

Description

(1) The figures show:

(2) FIG. 1:

(3) The Lewis glyco-epitope family, showing a subset of possible variants. The Le.sup.X glyco-epitopes carry fucose in an α1-3 linkage to the GlcNAc monosaccharide. A) sialylated, non-fucosylated GlcNAc; B) Lewis X (Le.sup.X) also called CD15 or SSEA-1; C) Sialyl Lewis X (sLe.sup.X).

(4) FIG. 2:

(5) IEF (isoelectric focusing) analysis shows increased sialylation of hAAT purified from glyco-optimized CAP cells stably, recombinantly expressing hAAT and ST3Gal4 or ST6Gal1 compared to hAAT purified from non-engineered hAAT expressing CAP cells. 5 μg of affinity purified hAAT per lane were subjected to isoelectric focusing. Different time points during pool generation are shown. Samples: CAP-hAAT-ST3Gal4, hAAT from CAP cell stably expressing human AAT as well as sialyltransferase ST3Gal4, CAP-hAAT-ST6Gal1, hAAT from CAP cell stably expressing human AAT as well as sialyltransferase ST6Gal1. Plasma derived hAAT (Prolastin), hAAT from non-glyco-optimized CAP cells, and desialylated hAAT from CAP cells served as controls.

(6) FIG. 3:

(7) Comparative lectin blot analysis of recombinant AAT reveals that a decreased amount of fucosylation correlates with increased amounts of sialylation. Purified human rAAT from either wild-type CAP cells, CAP-ST3Gal4 cells or CAP-ST6Gal1 cells were separated by SDS-PAGE, blotted on nitrocellulose membrane and detected by specific lectins. The corresponding densitometrical analysis (B) was normalized on the AAT protein content in the matching western blot. The Erythrina crista-galli lectin (ECL-lectin) analysis detects free galactoses on N-glycans which indicates incomplete sialylation. α1-3 linked fucose is detected by Lotus tetragonolobus agglutinin (LTA).

(8) FIG. 4:

(9) FACS analysis of cell surface glycoproteins of glyco-optimized CAP cells stably expressing ST3Gal4 or ST6Gal1 in comparison to non-engineered CAP cells reveals that overexpression of one of these two sialyltransferases not only increases the degree of sialylation on the majority of expressed glycoproteins. It also decreases the amount of antennary fucose on N-glycan structures, resulting in a reduced amount of Lewis.sup.X structures.

(10) FIG. 5:

(11) Increasing amounts of sialic acid (NANA) and reduced amounts of fucose (Fuc) in hAAT purified from CAP cells stably expressing either ST3Gal4 or ST6Gal1 compared to hAAT purified from cell culture supernatant from non-engineered CAP cells. Plasma derived hAAT (Prolastin) shown as control. Monosaccharide analysis was performed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC PAD).

(12) FIG. 6:

(13) Comparative lectin blot analysis of recombinant C1-Inhibitor reveals that an increase in sialylation correlates with a decrease in antennary fucosylation. Purified C1-Inhibitor from either wild-type CAP cells, CAP-ST3Gal4 cells or CAP-ST6Gal1 cells were separated by SDS-PAGE, blotted on nitrocellulose membrane and detected by specific lectins. The corresponding densitometrical analysis (B) was normalized to the C1-Inh protein content in the matching western blot. The Erythrina crista-galli lectin (ECL-lectin) analysis (A and B) detects free galactoses on N-glycans which indicates incomplete sialylation. α1-3 linked fucose is detected by Lotus tetragonolobus agglutinin (LTA).

(14) FIG. 7:

(15) MS-MS analysis of recombinant C1-Inhibitor: PNGase F released permethylated N-glycans from purified C1-Inhibitor either from wild-type CAP cells, from CAP-ST3Gal4 cells or from CAP-ST6Gal1 cells were analyzed by MALDI TOF/TOF. Only the signal at 3196.8 in MS1 of wildtype derived C1-Inhibitor (A) contained two fucose residues and the characteristic fragmentation pattern for antennary fucose (M/z 638, 505, 260; D) in MS2. In CAP-ST3Gal4 (B) and CAP-ST6Gal1 (C) the signal at 3196.8 could not be detected, also no other signal of MS1 contained a fragmentation pattern of antennary fucose in MS2.

(16) The present invention will be further illustrated in the following examples without being limited thereto.

EXAMPLES

(17) Experimental Procedures:

(18) Cell Culture and Fermentation.

(19) The permanent human amniocyte cell line CAP 1D5 was cultured in suspension, either in chemically defined, animal component free CAP-CDM medium (CEVEC Pharmaceuticals, Germany) supplemented with 6 mM stable glutamine (Biochrom, Germany), or in serum free PEM media (Life Technologies) supplemented with 4 mM stable glutamine (Biochrom, Germany). CAP cells were cultivated at 37° C. in shaker flasks (Corning, #431143, 125 mL (25 mL wv) or #431252, 3000 mL (1000 mL wv)) at 5% CO.sub.2, and 185 rpm. During fermentation, CAP cells were fed at d3, d5, and d7 with 10% CAP-CDM feed solution (CEVEC Pharmaceuticals, Germany) and 4 mM stable glutamine (Biochrom, Germany).

(20) Cloning.

(21) For the generation of CAP cell lines stably expressing ST3Gal4 or ST6Gal1, the cells were nucleofected with the corresponding nucleic acid constructs. Table 1 lists all cell lines created for this project.

(22) For designing the ST3Gal4 cDNA, sequence information of the precursor protein and mature protein was based on the database entry UniProt Q11206 (SEQ ID NO: 1). For cloning, a ClaI restriction site and a Kozak sequence were added 5′ of the start codon of the human ST3Gal4 cDNA and an EcoRV restriction site was added 3′ of the stop codon to be inserted between the ClaI and EcoRV restriction sites in the pStbl-Neo-CMV-MCS(−) vector resulting in the expression plasmid pStbl-Neo-CMV-ST3Gal4. This vector contains a CMV promoter driving the expression of the gene of interest, followed by an SV40 intron for improved, splicing-mediated mRNA transport and a multiple cloning site for the insertion of the gene of interest. The selection marker is driven by the human ubiquitin (UbC) promoter. cDNA synthesis was performed at GeneArt (Germany, Life Technologies).

(23) TABLE-US-00001 TABLE 1 Stable cell lines used in the present invention. overexpression of the Cell line rec. protein sialyltransferase(s) CAP / / CAP-AAT AAT / CAP-AAT-ST3Gal4 AAT ST3Gal4 CAP-AAT-ST6Gal1 AAT ST6Gal1 CAP-C1 Inh C1 Inh / CAP-C1 C1 Inh ST3Gal4 Inh-ST3Gal1 CAP-C1 C1 Inh ST6Gal1 Inh-ST3Gal4

(24) For designing the ST6Gal1 cDNA, sequence information of the precursor protein and mature protein was based on the database entry UniProt P15907 (SEQ ID NO: 2). For cloning, a ClaI restriction site and a Kozak sequence were added 5′ of the start codon of the human ST6Gal1 cDNA and an EcoRV restriction site was added 3′ of the stop codon to be inserted between the ClaI and EcoRV restriction sites in the pStbl-Neo-CMV-MCS(−) vector resulting in the expression plasmid pStbl-Neo-CMV-ST6Gal1. cDNA synthesis was performed at GeneArt (Germany, Life Technologies).

(25) Nucleofection and Pool Generation.

(26) Nucleofection was performed using a Nucleofector II (LONZA) with the appropriate Nucleofector Kit (KitV) according to the manufacturer's protocol. Briefly, during exponential growth phase of the culture 1×10.sup.7 cells were harvested via centrifugation (150 g for 5 min) and re-suspended in 100 μl complete Nucleofector solution and mixed with a total of 5 μg plasmid. Nucleofection was performed using the X001 program. After the pulse, cells were recovered in 12 ml complete cell culture media in a 125 ml shaking flask. The cells were cultured as before at 37° C., 5% CO.sub.2, and 185 rpm.

(27) 72 to 96 h post-nucleofection cells were selected with 200 μg/ml neomycin in order to generate stable pools.

(28) Western Blot Analysis.

(29) Purified protein solutions were separated on a NuPAGE Novex 4-12% Bis-Tris Gel under reducing conditions, according to the manufacturer's instructions. The separated proteins were transferred via a Blot Module (Invitrogen) (30 V for 60 min at RT) onto an Amersham Hybond ECL membrane (100 V for 60 min at RT). The membrane was blocked for 1 h at RT with PBSTB (phosphate-buffered saline, pH=7.4, supplemented with 0.1% Tween 20 and 1% BSA). Afterwards, the membrane was incubated with the specific horseradish peroxidase (HRP)-labeled antibody diluted in PBSTB. After washing the membrane with PBST (phosphate-buffered saline pH=7.4 supplemented with 0.1% Tween 20), the proteins were detected using the Pierce ECL WB Substrate Kit via a chemiluminescence detector (INTAS).

(30) Lectin Immunoblotting.

(31) Lectins are proteins that bind specific carbohydrate structures. Biotin-coupled lectins can therefore be used to analyze N-linked glycans. Erythrina crista-galli (ECL) lectin detects β1-4 linked terminal galactose on N-linked glycans, Sambucus nigra agglutinin (SNA) preferentially binds to α2,6-linked sialic acid, whereas Maackia amurensis lectin (MAL) preferentially binds to α2,3-linked sialic acids. α1-3 linked fucose is detected by Lotus tetragonolobus agglutinin (LTA) and Aleuria aurantia lectin (AAL) detects α1-2-, -3, or -6 linked fucose.

(32) Purified protein solutions from parental CAP cells with or without co-expression of ST3Gal4 and/or ST6Gal1 were separated as described above and blotted onto Amersham Hybond ECL nitrocellulose membrane (GE healthcare). The membrane was blocked for 1 h at RT with PBSTB (phosphate-buffered saline, pH=7.4, supplemented with 0.1% Tween 20 and 1% BSA). Afterwards, the membrane was incubated with the lectin diluted in PBSTB. After washing the membrane with PBST (phosphate-buffered saline, pH=7.4, supplemented with 0.1% Tween 20), the membrane was stained with streptavidin-coupled horseradish peroxidase (HRP) for 1 h at RT (diluted in PBSTB). The HRP signal was amplified using anti-streptavidin IgG and anti IgG-HRP. The proteins were detected using the Pierce ECL WB Substrate Kit via a chemiluminescence detector (INTAS).

(33) Isoelectric Focusing (IEF) Analysis.

(34) Isoelectric focusing (IEF) was performed in order to analyze the isoelectric point (pI) of rhAAT purified from CAP cells expressing rhAAT with or without additional expression of ST3Gal4 or ST6Gal1. The degree of sialylation correlates with a given proteins acidity and, therefore, with its pI. IEF analysis was done according to the manufacturers protocol (Invitrogen). Briefly, 5 μg of purified protein were loaded on pH 3-7 gels and subjected to electrophoresis (1 h 100 V, 1 h 200 V, 30 min 500 V). Proteins were stained with SimplyBlue SafeStain according to the manufacturer's protocol (Invitrogen).

Example 1

(35) Significantly Reduced Amount of Lewis.sup.X Structures on hAAT Protein Purified from CAP-ST3Gal4 or ST6Gal1 Cells.

(36) α1-Antitrypsin (AAT) is a protease inhibitor belonging to the serpin superfamily. AAT is a potent inhibitor of serine proteases, in particular neutrophil elastase. AAT is a 52 kDa glycoprotein carrying 3 N-glycosylation sites.

(37) Cells of the human amniocyte cell line CAP already stably expressing human AAT were additionally stably transfected with a plasmid encoding either the sialyltransferase ST3Gal4 to achieve an increase in 2,3-linked sialylation of terminal galactose of N-glycans or the sialyltransferase ST6Gal1 to achieve an increase in 2,6-sialylation of terminal galactose of N-glycans.

(38) Enhanced 2,3- or 2,6-sialylation upon overexpression of sialyltransferase ST3Gal4 or ST6Gal1 were determined by isoelectric focusing (IEF) analysis of purified hAAT (FIG. 2).

(39) As the backbones of the different rhAAT (recombinant hAAT) are identical, changes in the IEF indicate changes in the sialic acid content. Recombinant hAAT expressed in CAP cells with additional expression of ST3Gal4 results in a modified rhAAT which shifts significantly towards an acidic pI indicating an increased extent of sialylation; rhAAT expressed in parental CAP cells overexpressing ST6Gal1 also shifts towards a more acidic pI but to a lower degree (FIG. 2). This is probably due to the different substrate affinities of ST3Gal4 and ST6Gal1. ST6Gal1 catalyzes sialylation of the primary branches of N-glycans, whereas ST3Gal4 catalyzes the sialylation of the primary branches of N-glycan as well as the additional branches of tri- and tetra-antennary N-glycans. Therefore, ST3Gal4 has more acceptor substrate available than ST6Gal1.

(40) This result could be confirmed via lectin blot analysis (FIG. 3). Increased amounts of α2,3- or α2,6-sialylation upon overexpression of sialyltransferase ST3Gal4 or ST6Gal1 were determined via Erythrina crista-galli (ECL) lectin blot analysis. ECL lectin detects β1-4 linked terminal galactose on N-linked glycans. Therefore, a diminished signal in the ECL blot correlates to an increased amount of sialylation. Purified AAT from control CAP cells expressing AAT shows a clear signal in the ECL lectin blot, proving an incomplete sialylation. AAT derived from ST6Gal1 or ST3Gal4 overexpressing CAP cells, showed a strong reduction or complete absence of the ECL signal, indicating that only minimal amounts of unsialylated β1-4 linked galactose on the N-linked glycan exist in these preparations (FIG. 3).

(41) Remarkably, the degree of antennary fucose (Lewis.sup.x antigen) is reduced on rhAAT upon co-expression of ST6Gal1 or ST3Gal4 as proven by the reduced signal intensity in the Lotus tetragonolobus agglutinin (LTA Lectin) blot analysis in FIG. 3. Lotus tetragonolobus agglutinin (LTA) specifically detects the α1-3 linked antennary fucose. Therefore, overexpression of the sialyltransferases ST3Gal4 and ST6Gal1 is an unexpected but appropriate way to significantly reduce the amount of the unwanted and potentially immunogenic Lewis.sup.x structures on N-linked glycans.

Example 2

(42) FACS Analysis of Glycoproteins on the Cell Surface of CAP Cells Expressing ST3Gal4 or ST6Gal1.

(43) In order to determine the degree of fucosylation of glycoproteins on the cell surface with increased degree of sialylation by overexpression of sialyltransferases, flow cytometry (FACS) analyses was performed (FIG. 4).

(44) CAP-hAAT-ST3Gal4 or CAP-hAAT-ST6Gal1 cells were stained with different antibodies and lectins to analyze sugar epitopes on the surface of the cells. Typically, 1×10.sup.7 cells were centrifuged for 10 min at 140×g and re-suspended into 100 μl PBS/BSA. 10 μl (10.sup.6 cells) were mixed with 10 μl of antibody or lectin (1 mg/ml; FITC conjugated or DIG coupled in combination with a FITC coupled anti-DIG antibody) and 90 μl PBS/BSA were added. After 10 min at 4° C., the cells were washed with PBS/BSA. Cell pellets were re-suspended into 500 μl PBS/BSA and subjected to FACS analysis on a Becton Dickinson FACSCalibur flow cytometer. Dead cells were identified and excluded by staining with propidium iodide. Typically, 30000 events were counted and analyzed. FITC or PE stained cells were graphically overlaid with unstained cells.

(45) FIG. 4 shows parental CAP-hAAT cells or CAP-hAAT cells stably expressing α2,3 (CAP-hAAT+ST3Gal4) or α2,6 (CAP-hAAT+ST6Gal1) sialyltransferase, respectively. Cells were either stained with lectins specific for α2,3 coupled neuraminic acid, MAA (Maackia amurensis agglutinin), or α2,6 coupled neuraminic acid residues, SNA (Sambucus nigra agglutinin), or with LTA (Lotus tetragonolobus agglutinin) recognizing α1,3 linked fucose residues (Le.sup.X). Parental CAP-hAAT, HL60 (Le.sup.X pos.), Le.sup.X negative HEK293 cells (Le.sup.X neg.) as well as cells incubated with the FITC coupled anti-DIG antibody only are shown as controls.

(46) As expected, overexpression of α2,3- or α2,6-sialyltransferase increases the respective coupled neuraminic acid residues on the N-glycans of cell surface glycoproteins. Interestingly, expression of ST3Gal4 or ST6Gal1 reduces the amount of the non-preferred Le.sup.X structures on glycoproteins on the cell surface, as indicated by significantly reduced staining with lectin LTA.

Example 3

(47) Monosaccharide Analysis of hAAT Expressed in CAP-ST3Gal4 or CAP-ST6Gal1 Cells by HPAEC PAD Analysis.

(48) In order to determine if the total amount of sialic acid and fucose of purified recombinant hAAT from CAP cells changes upon additional expression of the sialyltransferases ST3Gal4 or ST6Gal1, a monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC PAD) was performed.

(49) FIG. 5 shows the relative amounts of sialic acid and fucose as determined by monosaccharide analysis via HPAEC-PAD in CAP-hAAT cells either stably transfected with a plasmid encoding ST3Gal4 or ST6Gal1.

(50) FIG. 5 reveals that by expressing human sialyltransferases, sialic acid content is elevated, whereas the amount of fucose per rhAAT molecule is significantly reduced relative to an un-transfected control, indicating that the amount of the non-preferred Le.sup.X or sialyl-Le.sup.x structure is reduced upon overexpression of sialyltransferases. Of note in this experiment, the total amount of fucose was determined without differentiating between antennary fucose residues (α1-3 linked fucose) and core fucose (α1-6 linked fucose) of N-glycans. As additional expression of sialyltransferases only affects the amount of antennary α1-3 linked fucose but not the relatively abundant core fucose, the determined overall reduction of fucose upon overexpression of sialyltransferases of 25% (ST6Gal1) and 30% (ST3Gal4) indicates a pronounced reduction in the absolute amount of antennary α1-3 linked fucose.

(51) The overall reduction in fucose residues is very surprising as the distal GlcNAc from sialylated complex N-glycans (NeuAc(α1->4)Gal(ß1-4)GlcNAc-R), which will be increased upon overexpression of ST3Gal4 and/or ST6Gal1, is a substrate for the fucosyltransferases Fut5, Fut6, and Fut7. Therefore, overexpression of sialyltransferases as ST3Gal4 should rather result in an increase in sialyl-Lewis.sup.x structures than an overall decrease in fucose residues.

Example 4

(52) Reduced Amount of Lewis.sup.x Structures on hC1 Inhibitor Protein Purified from CAP-ST3Gal4 or ST6Gal1 Cells.

(53) Cells of the human amniocyte cell line CAP-hC1 Inh were stably transfected with a plasmid encoding either the sialyltransferase ST3Gal4 to achieve increased α2,3-linked sialylation of terminal galactose of N-glycans or sialyltransferase ST6Gal1 to achieve increased α2,6-sialylation of terminal galactose of N-glycans.

(54) Increased amounts of α2,3- or α2,6-sialylation upon overexpression of sialyltransferase ST3Gal4 or ST6Gal1 were determined via Erythrina crista-galli (ECL) lectin blot analysis. ECL lectin detects β1-4 linked terminal galactose on N-linked glycans. As shown in FIG. 6, overexpression of ST3Gal4 results in an increased sialylation of the N-linked glycans in comparison to C1 Inh purified from cell culture supernatant from non-glycomodified CAP cells. Overexpression of ST6Gal1 also results in an increased sialylation of terminal galactose on N-linked glycans in comparison to the non-glycomodified C1-Inh, although the effect is not as pronounced as upon overexpression of ST3Gal4. This is most likely due to the different substrate affinities of ST3Gal4 and ST6Gal1 as explained in Example 1.

(55) The amount of antennary α1-3 linked fucose in the different C1 Inh protein preparations (CAP control, CAP-ST3Gal4, CAP-ST6Gal1) was determined by the Lotus tetragonolobus agglutinin (LTA) blot analysis. FIG. 6 reveals that the increased sialylation upon overexpression of ST6Gal1 or ST3Gal4 correlates with a reduced amount of antennary fucosylation proven by the decreased signal intensity in the LTA lectin blot analysis.

(56) These results were confirmed by MS-MS analysis of N-glycans from C1 Inh derived in CAP control cells, CAP-ST3Gal4, or CAP-ST6Gal1 cells (FIG. 7). PNGaseF released permethylated N-glycans from purified C1-Inhibitor either from control CAP cells, from CAP-ST3Gal4 cells or from CAP-ST6Gal1 cells were analyzed by MALDI TOF/TOF. Only the signal at 3196.8 in MS1 in wildtype derived C1-Inhibitor (FIG. 7 A) contained two fucose residues and the characteristic fragmentation pattern for antennary fucose (M/z 638, 505, 260; D) in MS2. In CAP-ST3Gal4 and CAP-ST6Gal1 (FIGS. 7 B and C) the signal at 3196.8 could not be detected. Moreover, no other signal of MS1 contained a fragmentation pattern of antennary fucose in MS2.

(57) The present invention relates to the following amino acid sequences.

(58) TABLE-US-00002 Human ST3Gal4 SEQ ID NO: 1 MVSKSRWKLLAMLALVLVVMVWYSISREDRYIELFYFPIPEKKEPCLQGE AESKASKLFGNYSRDQPIFLRLEDYFWVKTPSAYELPYGTKGSEDLLLRV LAITSSSIPKNIQSLRCRRCVVVGNGHRLRNSSLGDAINKYDVVIRLNNA PVAGYEGDVGSKTTMRLFYPESAHFDPKVENNPDTLLVLVAFKAMDFHWI ETILSDKKRVRKGFWKQPPLIWDVNPKQIRILNPFFMEIAADKLLSLPMQ QPRKIKQKPTTGLLAITLALHLCDLVHIAGFGYPDAYNKKQTIHYYEQIT LKSMAGSGHNVSQEALAIKRMLEMGAIKNLTSF Human ST6Gal1 SEQ ID NO: 2 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKS LGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDS SSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNV SMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGR EIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYN EGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQM PWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKT DVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPG FRTIHC Human AAT SEQ ID NO: 3 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKI TPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEI LEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKL VDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKEL DRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGM FNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAP LKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIE QNTKSPLFMGKVVNPTQK Human C1 Inh SEQ ID NO: 4 MASRLTLLTLLLLLLAGDRASSNPNATSSSSQDPESLQDRGEGKVATTVI SKMLFVEPILEVSSLPTTNSTTNSATKITANTTDEPTTQPTTEPTTQPTI QPTQPTTQLPTDSPTQPTTGSFCPGPVTLCSDLESHSTEAVLGDALVDFS LKLYHAFSAMKKVETNMAFSPFSIASLLTQVLLGAGENTKTNLESILSYP KDFTCVHQALKGFTTKGVTSVSQIFHSPDLAIRDTFVNASRTLYSSSPRV LSNNSDANLELINTWVAKNTNNKISRLLDSLPSDTRLVLLNAIYLSAKWK TTFDPKKTRMEPFHFKNSVIKVPMMNSKKYPVAHFIDQTLKAKVGQLQLS HNLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKLEMSKFQPTLLTLPR IKVTTSQDMLSIMEKLEFFDFSYDLNLCGLTEDPDLQVSAMQHQTVLELT ETGVEAAAASAISVARTLLVFEVQQPFLFVLWDQQHKFPVFMGRVYDPRA