Abstract
Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.
Highlights
Polysialic acid (PSA) plays a crucial role on the surface of eukaryotic neuronal cells and neuro-invasive pathogens like Neisseria meningitidis
Using primers based on the sequences of M. haemolytica A1 capsule biosynthesis genes wzf and phyB, a region of the M. haemolytica A2 genome was amplified and the sequence was analyzed using primer walking
The interest in using such PSTs for potential therapeutic applications requires that the enzyme of choice have qualities that are compatible with modification of high value therapeutic proteins, such as high purity and easy removal after modification
Summary
Polysialic acid (PSA) plays a crucial role on the surface of eukaryotic neuronal cells and neuro-invasive pathogens like Neisseria meningitidis. In mammals PSA is presented as a homopolymer of a2,8-linked N-acetyl-neuraminic acid (Neu5Ac) residues on a small number of proteins of which the major carrier is the neural cell adhesion molecule (NCAM). It has been shown that the PSA on NCAM modulate cell-cell interactions through antiadhesive properties during development (reviewed in [1]). The NCAM molecule has been implicated in numerous normal and pathological processes, including mammalian development, neuronal plasticity, and tumour metastasis [2]. PSA is found on CD36 [3], as well as on the recently described SynCAM [4]. The PSA portion of NCAM was shown to have a very important role in the brain since removal of the polysialylation resulted in severe brain defects and precocious death [5,6]. Changes to the amount and chain length of PSA may influence psychiatric disorders through impaired binding to brainderived neurotrophic factor and dopamine, which are linked to schizophrenia and other psychiatric disorders, such as depression and bipolar disorder [7]
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