Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

Abstract

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

Engineering of complex protein sialylation in plants

“…These activities of protein-independent polySia have great potential for the development of novel carbohydrate-dependent therapeutics. Likewise, improving the efficacy of therapeutic proteins (e.g., erythropoietin and buturylcholinesterase) using polySia was demonstrated by chemical conjugation and in vitro approaches (7,8,44). Clinical trials of polySia-conjugated recombinant proteins are ongoing, indicating the pharmaceutical relevance of polySia (reviewed in ref.…”

“…1, 2, 5, and 6). Notably, chemical or in vitro polySia engineering has made a substantial impact on pharmacokinetic properties of recombinant glycoprotein therapeutics (7,8). These examples demonstrate a manifold functional impact of polySia and highlight the great potential for the development of novel polySia-dependent therapeutics.…”

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

“…Natural and recombinant forms of this protein 4 have three N-glycosylation sites where the predominant glycoforms are bi-antennary sialylated structures [6][7][8]. Both of these glycoproteins are being produced for therapeutic purposes and modification of their glycans may alter their biological activity [9] and their pharmacodynamics [10].…”

Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

“…Polymerases are being investigated for various roles in enzymatic (and chemoenzymatic) syntheses (e.g. (4,(51)(52)(53) where the goal is to produce "glycoproducts" with defined chemical and physical characteristics. Understanding the structure and function of multidomain polymerases will be essential for their successful application in glycoengineering efforts.…”