GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli

Self Cite

115

120

Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by zinc finger nuclease targeting of a key gene in Oglycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific Oglycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.apolipoproteins | angiopoietin-like proteins | genetic engineering | glycoproteins T he GALNT2 gene involved in O-glycosylation has been associated with aberrant serum levels of triglyceride (TG) and highdensity lipoprotein cholesterol (HDL-C) by several genome-wide association studies (GWAS) (1, 2). A direct functional role of this gene was obtained by transient knockdown and overexpression of galnt2 in mouse liver, which resulted in increased and lowered plasma HDL-C, respectively (3). GALNT2 is a member of the largest family of homologous glycosyltransferase isoforms (up to 20) catalyzing the same glycosidic linkage (GalNAcα1-O-Ser/Thr) and initiating protein O-glycosylation (4, 5). These isoforms have overlapping but distinct peptide substrate specificities and identifying essential unique functions for individual GalNAc-T isoforms has been notoriously difficult. In search of putative functions of GALNT2 in lipid metabolism, we previously screened a number of known and predicted O-glycoproteins with roles in lipid metabolism for O-glycosylation by the encoded GalNAc-T2 enzyme and identified ANGPTL3 (6). We found that site-specific O-glycosylation of a Thr residue adjacent to the proprotein convertase (PC) processing site in ANGPTL3 that activates this lipase inhibitor was performed only by the GalNAc-T2 isoform. More recently, a heterozygous mutation in GALNT2 resulting in slightly reduced kinetic properties of the encoded GalNAc-T2 enzyme was found in two probands with elevated HDL and reduced TG (7). ...

Section: Resultssupporting

confidence: 76%

Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells

Schjoldager

Vakhrushev²,

Kong³

et al. 2012

Self Cite

115

120

“…Importantly, this technology allows one to rapidly optimize the physical, biological, and pharmacological properties of a protein conjugate through recombinant-based protein chemistry that is unrestricted in site of conjugation. This constitutes a significant advance relative to conventional conjugation methods that are restricted by the native 20 amino acids (16)(17)(18). For example, the modification of lysines by electrophiles that is widely used in research settings results in mixtures of positional isomers even when the stoichiometry is controlled to provide one PEG per protein.…”

Section: Discussionmentioning

confidence: 99%

Optimized clinical performance of growth hormone with an expanded genetic code

Cho

Daniel²,

Buechler

et al. 2011

184

187

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.protein engineering | endocrinology | bio-better

“…PEGylation of proteins relies on chemical conjugation of PEG chains to free amino groups or engineered Cys residues on the protein, which can lead to heterogeneously modified proteins whose activity can be adversely affected (3). An alternative method for site-specific PEGylation of proteins has been termed GlycoPEGylation, whereby PEG chains conjugated to sialic acid residues are transferred onto unoccupied, natural glycosylation sites on therapeutic proteins (4). Despite the low immunogenicity of PEG, the production of anti-PEG antibodies and the accumulation of nonmetabolized PEG in tissues remain concerns (5) and warrant exploration of alternative protein modification strategies.…”

mentioning

confidence: 99%

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Lindhout

Iqbal

Willis

et al. 2011

103

The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II α2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis α2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.glycosyltransferase | glycosylation