Cell-contact mediated modulation of the sialylation of contactinhibin

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated -glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularily, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.     

Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co‐expression of BChE with a novel gene‐stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N‐glycans. The N‐glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma‐derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER‐typical oligomannosidic structures. Biochemical analyses and live‐cell imaging experiments indicated that impaired N‐glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic‐reticulum‐derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in‐depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

Alteration of the sialylation pattern of the murine tooth germ after ethanol exposure

BACKGROUND: Ethanol consumption during pregnancy leads to changes in murine dental morphogenesis, dental size, cellular differentiation, enamel mineralization, and delayed eruption. It has been proposed that glycoproteins play a role during embryonic dental development that may determine the dental morphological pattern and extracellular matrix secretion. O-glycosylation and sialylation appear to actively participate in the differentiation and maturation processes. Because glycosylation may be affected by teratogens that can alter the maturation of several organisms, in this work we describe the main modifications of the sialylation pattern in prenatal day (PD) 18.5 murine tooth germs exposed to ethanol. METHODS: Pregnant female mice were divided into groups that were given 15% or 20% ethanol solutions, or water as a control. The histochemistry of tooth germs from PD 18.5 fetuses was revealed with lectins specific for sialic acid (Neu5Ac), such as Sambucus nigra (SNA), Maackia amurensis (MAA), and Machrobrachium rosenbergii (MRL), and for sialylated-O-glycosidically linked glycans, such as Amaranthus leucocarpus (ALL). RESULTS: The basement membrane, preameloblasts, inner-enamel epithelium, preodontoblasts, and subodontoblastic cells of the test groups showed changes in labeling according to the 4 lectins used. Intranuclear staining was observed with SNA (specific for Neu5Acalpha2,6Gal/GalNAc) in the control group, but this was reduced in the test groups. The nuclei of dental papillary cells under the experimental conditions were stained with MAA (Neu5Acalpha2,3Gal). CONCLUSIONS: Dental development involves different types of sialylated O-glycosidically linked glycans that are likely to regulate cell-to-cell and cell-to-matrix interactions. Our results suggest that ethanol consumption during pregnancy alters the sialylation pattern during murine dental morphogenesis.     

Effect of Bcl‐xL overexpression on sialylation of Fc‐fusion protein in recombinant Chinese hamster ovary cell cultures

The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl‐x_L overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc‐fusion protein, which were derived from DUKX‐B11 and DG44, respectively, were engineered to have regulated Bcl‐x_L overexpression using the Tet‐off system. For both cell lines, Bcl‐x_L overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc‐fusion protein titer increased by Bcl‐x_L overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl‐x_L overexpression, the sialylation of Fc‐fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl‐x_L overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl‐x_L overexpression in rCHO cell culture increased Fc‐fusion protein production and also reduced the impairment of sialylation of Fc‐fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1133–1136, 2015     

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.     

Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression

Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells.     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

The sialylation of gonococcal lipopolysaccharide by host factors: A major impact on pathogenicity

The resistance of gonococci in most patients to complement mediated killing by human serum is due to sialylation of their lipopolysaccharide (LPS) which prevents bactericidal antibody from reacting with target sites. Two of the host factors responsible are: cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA), a well-known sialylating agent, and another factor which enhances the transfer of sialyl groups from CMP-NANA to LPS catalysed by a gonococcal sialyltransferase. The bacterial determinant of resistance is a conserved LPS component of about 4.5 kDa which is sialylated at a terminal Gal beta 1-4GlcNAc site on its side chain. The sialylated LPS forms a surface coat which is stainable by ruthenium red and connected with previously described 'capsules'. These observations sparked off an explosion of research. Recent publications show that sialylation of LPS by CMP-NANA affects additional important aspects of gonococcal pathogenicity, notably interactions with antibodies and phagocytes, and rendering the gonococcal surface more 'host-like'. Also, the observations have prompted an examination of LPS from some other pathogens for the presence of sialyl groups with positive results for Neisseria meningitidis and Haemophilus influenzae.     

Effects of α-benzyl-α-bromo-malodinitrile on the primary electron acceptor of Photosystem II in spinach chloroplasts

Reactions at the reducing side of Photosystem II in spinach chloroplasts are modified by α-benzyl-α-bromo-malodinitrile (BBMD).On addition of 50 μM BBMD to chloroplasts the following phenomena can be observed: (1) electron flow to an acceptor like 2,6-dichlorophenolindophenol is partly deflected to electron flow to oxygen; (2) the electron flow to oxygen is carbonyl cyanide m-chlorophenylhydrazone sensitive but 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive; (3) variable fluorescence is abolished but basal fluorescence is not altered; (4) a strong photobleaching of carotenoids is induced. BBMD seems a very efficient acceptor for electrons from the primary electron acceptor of Photosystem II, resulting in a BBMD-mediated electron transport from this primary acceptor to oxygen.On pretreatment of chloroplasts with 50 μM BBMD the effects are different; (1) electron flow to 2,6-dichlorophenolindophenol, ferricyanide, or NADP is almost completely inhibited and is not restored by addition of artificial electron donors: (2) no electron flow to oxygen is observable unless BBMD again is added to reaction media; (3) no variable fluorescence is observable but basal fluorescence is not affected; (4) there is no photobleaching of carotenoids unless BBMD again is added; (5) no reduction of C-550 can be recorded. Pretreatment of chloroplasts with BBMD seems to induce an intense cycling of electrons around Photosystem II and only anew added BBMD can interrupt this cycling.     

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.     

Effect of site-specific PEGylation on the fibrinolytic activity,immunogenicity, and pharmacokinetics of staphylokinase

The bacterial plasminogen-activator staphylokinase （Sak） is a promising thrombolytic agent for treating acute myocar- dial infarction. To effectively reduce the immunogenicity of Sak while maintaining its fibrinolytic activity, site-specific PEGylation was performed in the present study. The che- moselective cysteine PEGylation site was selected within an immunodominant region （amino acid residues 71-87） using an in sUico approach. The PEGylated Sak variants pre- pared in this study showed a purity of 〉97.0%. PEGylation at Position 80 resulted in a Sak variant Sak（E80C-PEG） which has similar fibrinolytic activity and thermostability compared with the native recombinant staphylokinase （r-Sak）. The immunogenicity of Sak（E80C-PEG） in guinea pigs was greatly reduced compared with the native r-Sak. Furthermore, preliminary pharmacokinetic results sug- gested that the plasma clearance of Sak（E80C-PEG） from the blood stream of rabbit was significantly decreased com- pared with that of r-Sak, resulting in a 2.8-fold increase of initial half-life and a 3.8-fold increase of systemic availabil- ity. In summary, these results demonstrated that site-specif- ic PEGylation yielded a novel Sak variant Sak（E80C-PEG） with remarkable advantages over the unmodified Sak.     

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.     

Effect of cold-storage of bean leaves on photosynthetic reactions of isolated chloroplasts. Inability to donate electrons to photosystem II and relation to manganese content

1.

1. Chloroplasts from bean leaves which have been aged in darkness at o °C do not carry out Hill reactions. They reduce NADP with reduced 2,6-dichlorophenolindophenol (DCIP) in a reaction that is insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). They reduce NADP with hydroquinone, p-phenylenediamine or benzidine, and reduce DCIP with hydroxylamine, 1,4-diphenylsemicarbazide, 1,5-diphenylcarbohydrazide or manganous ion in DCMU-sensitive reactions.     

Effect of culture conditions on the degree of sialylation of a recombinant glycoprotein expressed in insect cells

Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.     

Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The synthesis of the pentasaccharide, beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-beta-D-Galp-(1-->6)-[beta-D-Galf-(1-->4)]-D-GlcpNAc and the corresponding alditol, previously isolated by reductive beta-elimination of the mucins, is described. The key step was the 6-O-glycosylation of a easily accessible derivative of beta-D-Galf-(1-->4)-D-GlcpNAc with a beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-D-Galp donor using the trichloroacetimidate method. The beta-linkage was diastereoselectively obtained by the nitrile effect. The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal beta-D-Galp residues for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated product was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal (1-->3) linked galactopyranose.