Endosialidases: Versatile Tools for the Study of Polysialic Acid

Abstract

Polysialic acid is an α2,8-linked N-acetylneuraminic acid polymer found on the surface of both bacterial and eukaryotic cells. Endosialidases are bacteriophage-borne glycosyl hydrolases that specifically cleave polysialic acid. The crystal structure of an endosialidase reveals a trimeric mushroom-shaped molecule which, in addition to the active site, harbors two additional polysialic acid binding sites. Folding of the protein crucially depends on an intramolecular C-terminal chaperone domain that is proteolytically released in an intramolecular reaction. Based on structural data and previous considerations, an updated catalytic mechanism is discussed. Endosialidases degrade polysialic acid in a processive mode of action, and a model for its mechanism is suggested. The review summarizes the structural and biochemical elucidations of the last decade and the importance of endosialidases in biochemical and medical applications. Active endosialidases are important tools in studies on the biological roles of polysialic acid, such as the pathogenesis of septicemia and meningitis by polysialic acid-encapsulated bacteria, or its role as a modulator of the adhesion and interactions of neural and other cells. Endosialidase mutants that have lost their polysialic acid cleaving activity while retaining their polysialic acid binding capability have been fused to green fluorescent protein to provide an efficient tool for the specific detection of polysialic acid.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

“…Owing to the high specificity of depolymerases, it was suggested that enzyme-based capsular typing may be more useful than whole phage typing (Hsu et al 2013; Lin et al 2014). The analogous approach has been successfully applied to develop an endosialidase-based detection reagent for identification of polysialic acid-containing bacteria as well as eukaryotic cells (Jakobsson et al 2015). As a detection tool, these enzymes are used in a catalytically inactive form, capable to recognize and bind the substrate, but without its degradation.…”

A crucial role for polysialic acid in developmental interneuron migration and the establishment of interneuron densities in the mouse prefrontal cortex

“…Slices were preincubated for 30 min on ice in dissection buffer supplemented with 10 mM HEPES, 1× penicillin/streptomycin (Biochrom) and 50 μg/ml gentamycin (Sigma-Aldrich). Where indicated, slices were incubated with 4 μg/ml endosialidase (endo) (Stummeyer et al, 2005), which reliably and with high specificity removes polySia from the surface of living cells in culture and in tissues (Jakobsson et al, 2012). After transfer to Millicell cell culture inserts (PICM0RG50, Merck-Millipore) in six-well plates, slices were cultured for 1 day at 37°C and 5% CO 2 in growth medium 1 comprising Neurobasal medium (Life Technologies) containing 1× B27 supplement (Life Technologies), 32 mM D-glucose, 2 mM L-glutamine, 1× penicillin/streptomycin and 4 μg/ml endo, where indicated.…”

Structure and biochemical characterization of bacteriophage phi92 endosialidase

“…Folding of the trimeric endoNF involves auto-proteolytic release of its C-terminal chaperone domain in a Ser-Lys dyad reaction (Mühlenhoff et al, 2003;Schwarzer et al, 2007;Schulz et al, 2010a;Schulz and Ficner, 2011). The release of the chaperone domain increases thermal stability of endoNF (Schwarzer et al, 2007) and unmasks a polySia binding site located in the stalk domain conferring processivity to the enzyme (Schwarzer et al, 2009;Jakobsson et al, 2012).…”

“…About thirty genomes of E. coli K1-specific phages have been characterized so far and all contain a gene that encodes an endoN (Jakobsson et al, 2012). The tailspike of coliphage K1F (endoNF) serves as a paradigm for all these enzymes as its function, folding properties, and crystal structure in the apo-and substrate-bound form have been well characterized (Mühlenhoff et al, 2003;Stummeyer et al, 2005;Schwarzer et al, 2007Schwarzer et al, , 2009Schulz et al, 2010bSchulz et al, , 2010c.…”