Endoglycosidase H digestion of neuronal intracellular lectin-binding sites

Cerebellar Purkinje cells contain intracellular membrane systems that are highly enriched in concanavalin A (Con A)-binding sites and which may be involved in axonal and dendritic transport. We have attempted to characterize these Con A-binding sites in fixed slices of cerebellum by their susceptibility to endoglycosidase H (Endo H) digestion. Tissue slices labeled with Con A-peroxidase without prior Endo H digestion had moderately dense label in the molecular and granular layers with the heaviest label occurring in Purkinje cell somata. Endo H digestion of tissue slices produced little change in the pattern of Con A label in the molecular and granular layers, but the Con A label in Purkinje cell somata was removed except for small discrete patches. Electron microscopic examination of Purkinje cells from tissue slices not digested with Endo H showed Con A label in cisternal elements of the endoplasmic reticulum (ER), nuclear envelope, hypolemmal cisternae, and the Golgi complex. Presynaptic terminal smooth membrane cisternae and dendritic hypolemmal cisternae of Purkinje cells were also labeled with Con A-peroxidase. Endo H digestion removed Con A-binding sites in cisternae of the ER, somal and dendritic hypolemmal membrane system, and presynaptic terminal smooth membrane profiles. Con A-binding sites in the nuclear envelope and Golgi complexes of Purkinje cells were not removed by Endo H digestion. These results present ultrastructural evidence in support of biochemical studies which report the rough ER as the site of polymannose oligosaccharide addition to nascent polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)