Chemoenzymatic Synthesis of a Phosphorylated Glycoprotein (original) (raw)
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Molecular therapy. Methods & clinical development, 2017
Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.
Development of fully functional proteins with novel glycosylation via enzymatic glycan trimming
Journal of Pharmaceutical Sciences, 2009
Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N-linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an a-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. ß
In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins
Journal of Controlled Release, 2009
Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human α-galactosidase (rhαGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in αGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution.
ACS Macro Letters, 2016
Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. Unless otherwise reported, all reactions were performed under argon atmosphere. Removal of solvent in vacuo refers to distillation using a rotary evaporator attached to an efficient vacuum pump. Products obtained as solids or syrups were dried under high vacuum. 9-BBN dimer, BF3-diethyl ether and FL-NHS ester was purchased from Sigma Aldrich. All other chemicals used were obtained from Merck, India. Diethyl ether, petroleum ether (60−80 °C), ethyl acetate, dichloromethane, tetrahydrofuran, and dioxane were purchased from Merck, India; dried by conventional methods and stored in the glove box.Analytical thinlayer chromatography was performed on pre-coated silica plates (F254, 0.25 mm thickness); compounds were visualized by UV light or by staining with anisaldehyde spray. 1 H NMR spectra were recorded on Bruker Spectrometers (200 MHz, 400 or 500 MHz) and reported relative signals according to deuterated solvent used. 13 C NMR and DEPT spectra were recorded on Bruker Spectrometer (50, 100, or 125 MHz), and reported relative signals according to deuterated solvent used. 31 P NMR spectra recorded on Bruker Spectrometer (202.46 MHz) using an internal 85% aqueous H3PO4 as reference. FT-IR spectra were recorded on Perkin-Elmer FT-IR spectrum GX instrument by making KBr pellets. Pellets were prepared by mixing 3.0 mg of sample with 97.0 mg of KBr. (GPC) was performed on a VISKOTEK TDA 305-040 Triple Detector array refractive index (RI), viscometer (VISC), low angle light scattering (LALS), right angle light scattering (RALS) GPC/SEC module. Using these triple detection system absolute molecular weights was determined by using a dn/dc value of 0.059. Separations were achieved by three columns (T6000M, GENERAL MIXED ORG 300x7.8 MM) and one guard column (TGUARD, ORG GUARD COL 10x4.6 MM), 0.025 M LiBr in DMF as the eluent at 60 °C. GPC samples were prepared at concentrations of 5 mg/mL. A constant flow rate of 1 mL/min was maintained. System was calibrated by PMMA standards. Cell lines were purchased from ATCC and were maintained as per the manufacturer's instructions. Preparation of 9-BBN complex of Lysine L-lysine mono hydrochloride salt was neutralized by treatment with aq. NH3 solution in a round bottom flask for 30 min at 0˚C and then concentrated under vacuum to remove excess ammonia. This was directly used for 9-BBN complex formation reaction. To 150mL of methanol in a 250mL round bottom flask at room temperature under argon was added 1.05 eq. of 9-BBN dimer. The mixture was heated at reflux until the 9-BBN was completely dissolved (30 min) and to this solution 25 mmol of amino acid was added. The resultant reaction mixture was heated for additional 3 h until gas evolution ceased and the suspension became a clear homogenous solution. The methanol was removed on rotary evaporator and the residue dissolved in hot THF (100 mL), filtered and the filtrate was concentrated to afford a white gummy residue of 9-BBN-L-lysine complex. Excess of 9-BBN was removed by washing with hot hexane and then subjected to high vacuum for 1 h during which time it became an amorphous solid. This material was used without any further purification for the subsequent coupling reaction. Preparation of (allyl)-2,3,4,6-tetra-O-acetyl-β-D-mannopyranoside (1a) To a suspension of D-mannose (10g, 55.56 mmol) in glacial acetic acid (100 mL) and acetic anhydride (39.35 mL, 416.7 mmol)was added conc. H2SO4 (2 mL) drop wise and the reaction mixture was stirred at room temperature. After half an hour the reaction mixture became clear indicating completion of the reaction. The reaction mixture was dissolved in 200 mL of DCM and washed with cold water (3×300 mL) and then saturated bicarbonate solution to afford penta-O-acetyl-β-D-mannopyranoside without any acid impurities. The DCM solution was washed with brine followed by drying over sodium sulfate and removal of solvent by rotary evaporator to obtain completely pure penta-O-acetyl-β-D-mannopyranoside (Yield 21.5g; ~99%). Penta-O-acetyl-β-D-mannopyranoside (21.5g, 25.6 mmol) and allyl alcohol (2.09 mL, 30.7 mmol) in dry dichloromethane (50 mL) was placed in 250 mL round bottom flask which was covered by aluminium foil and fitted with a dropping funnel. The solution was cooled to 0˚C after which BF3.Et2O (31.1 mL, 219.2 mmol) was added drop wise over a period of 30 minutes. The reaction mixture was then stirred for an additional 2 h at 0˚C and then subsequently at room temperature for 12 h. Completion of reaction was monitored by TLC. The reaction mixture was diluted by dichloromethane and poured onto ice water while stirring. The organic layer was separated and washed successively with water, saturated sodium bicarbonate and brine. The organic layer was dried on Na2SO4, concentrated on rotary evaporator and the resulting residue was purified by column chromatography on silica gel using ethyl acetate-petroleum ether as eluent to get allyl-penta-O-acetyl-β-Dmannopyranoside (13.8g, 65%) as a crystalline solid.
PLoS ONE, 2010
Background: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the diN -acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo b-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. Methods/Principal Findings: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1D deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man 8 GlcNAc 2 , corresponding to ,70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc 1 Man 9 GlcNAc 2 and Man 9 GlcNAc 2 that corresponds to ,30% of total fOS. Conclusions/Significance: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.
Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy
Journal of inherited metabolic disease, 2001
The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease. The diseases frequently have a progressive neurodegenerative course. The therapeutic options for treating these diseases are relatively limited, and for the majority there are no effective therapies. The problem is further compounded by difficulties in delivering therapeutic agents to the brain. Most research effort to date has focused on strategies for augmenting enzyme levels to compensate for the underlying defect. These include bone marrow transplantation (BMT), enzyme replacement and gene therapy. An alternative strategy that we have been exploring is substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. The imi...
Biotechnology Journal, 2013
Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre-and post-exposure treatment. The presence and functional necessity of complex Nglycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. We aim to produce recombinant BChE (rBChE) in plants with a glycosylation profile that largely resembles the plasma-derived counterpart. rBChE was transiently co-expressed in the model plant Nicotiana benthamiana. Site-specific sugar profiling by mass spectrometry of secreted rBChE collected from the intercellular fluid (IF) revealed the presence of mono-and di-sialylated Nglycans, with overall glycosylation profile that is virtually identical to the plasma-derived orthologue. Increase in sialylation content of rBChE was acehived by the over-expression of an additional glycosylation enzyme that generates branched N-glycans, (i.e. GnTIV), which resulted in the production of rBChE decorated with a large fraction of tri-sialylated structures. Sialylated as well as non-sialylated plant-derived rBChE exhibit functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.
Enhanced glycosylation with mutants of endohexosaminidase A (endo A)
2008
Glycosylation of proteins is the most diverse form of posttranslational modification, and can play a key role in protein folding, [1] and can also crucially affect important protein properties. [2-6] However, since the biosynthesis of glycans is not under direct genetic control, glycoproteins are produced intracellularly as heterogeneous mixtures of glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. Access to pure single glycoforms of glycoproteins has now become a major scientific objective [7] since it is not only a prerequisite for more precise biological investigations into the different effects glycans have on protein properties, but also an important commercial goal in the field of glycoprotein therapeutics, which are currently marketed as heterogeneous mixtures of glycoforms. Access to single glycoforms of glycoproteins can be achieved by total synthesis of both glycan and polypeptide components, and some outstanding achievements in this area have recently been published. [8, 9] However, such A C H T U N G T R E N N U N G synthesis approaches are particularly arduous and do not realistically represent a practical approach that could be applied to widespread and large-scale glycoprotein production. Alternative approaches based on bioengineering of cell lines in order to optimise production of glycoproteins that bear particular oligosaccharide structures have also been reported [10, 11] and are being exploited commercially, though such approaches have no guarantee of complete glycan homogeneity. An alternative method for achieving homogeneous protein glycosylation involves the use of enzymatic catalysis, [12] and one particular class of enzyme that displays considerable synthesis potential in this respect comprises the endohexosaminidases. [13] Endohexosaminidases are a class of enzyme that specifically cleave the chitobiose core [GlcNAcbA C H T U N G T R E N N U N G (1-4)GlcNAc] of Nlinked glycans between the two N-acetyl glucosamine residues, and since they cleave this linkage they can also be used to selectively synthesise it. Two members of this class that have been demonstrated to display useful synthesis glycosylation activity are Endo M from Mucor hiemalis [14-17] and Endo A from Arthrobacter protophormiae. [18, 19] However, since these enzymecatalysed reactions are reversible, competitive product hydroly-[a] Dr.
An Endoglycosidase with Alternative Glycan Specificity Allows Broadened Glycoprotein Remodelling
Journal of the American Chemical Society, 2012
Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reveal that a bacterial endoglycosidase from Streptococcus pyogenes, EndoS, is complementary to other known endoglycosidases (EndoA, EndoH) used for current protein remodeling. It allows processing of complex-type N-linked glycans +/− core fucosylation but does not process oligomannose-or hybrid-type glycans. This biocatalytic activity now addresses previously refractory antibody glycoforms.