Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.

Abstract

Three prokaryotic-derived probes to identify and study the temporal expression of polysialosyl units in neuronal tissue were developed. A polyclonal antibody, a bacteriophage-derived endo-neuraminidase and an Escherichia coli K1 sialyltransferase are all specific for either recognizing or synthesizing poly(sialic acid) containing .alpha.-2,8-ketosidic linkages. Polysialosyl immunoreactivity with apparent MW values of 180,000-240,000 was specific for developing neuronal tissue; it was not detected in neonatal liver or kidney or in adult brain tissue. The developmentally regulated disappearance in poly(sialic acid) is consistent with the probes described here recognizing the polysialosyl carbohydrate units of a neuronal cell adhesion molecule (N-CAM). Treatment of brain extracts with a bacteriophage-derived endo-neuraminidase specific for .alpha.-2,8-linked polysialosyl units abolished the immunoreactivity. The material solubilized by endo-neuraminidase was isolated, reduced with borotritide, and shown to contain oligomers of sialic acid with 3-6 sialyl units. Treatment of the 3H-labeled oligosialic acid with exo-neuraminidase quantitatively converted the radioactivity to sialitol, establishing that the brain-derived oligomers were composed solely of sialic acid. A membranous sialyltransferase from E. coli K1 that can transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid) also recognized rat brain membranes, further substantiating the presence of poly(sialic acid) in rat brain. This was confirmed by using a mutant of E. coli K1 defective in the synthesis of poly(sialic acid) and could only transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid). Sialyl polymer synthesis was restored in the mutant when brain membranes were added as exogenous acceptor.