EXPRESSION CLONING AND CHARACTERIZATION OF NSIST, A NOVEL SULFOTRANSFERASE EXPRESSED BY A SUBSET OF NEURONS AND POSTSYNAPTIC TARGETS

Synapses are distinguished by localized concentrations of specific pro teins, many of which bear the marks of posttranslational processing su ch as glycosylation and sulfation. One strategy to elucidate this post translational tailoring is to identify the enzymes that create these m odifications. Monoclonal antibody 3B3 recognizes a carbohydrate-contai ning epitope expressed on dystroglycan and other constituents of Torpe do electric organ synaptic membranes. We used mAb 3B3 in an immunofluo rescence-based expression-cloning method and isolated a cDNA clone con ferring mAb-3B3 immunoreactivity to transfected COS cells. The deduced polypeptide has a predicted molecular weight of 51 kDa, a type II tra nsmembrane topology, and four potential N-linked glycosylation sites. The polypeptide, which we term NSIST (nervous system involved sulfotra nsferase), shows extensive, although not complete, homology to a chond roitin-8-sulfotransferase and limited homology to other sulfotransfera ses, In NSIST-transfected COS cells, (SO4)-S-35, incorporation and cho ndroitin-sulfate-like immunoreactivity are increased. In vivo, NSIST o ccurs as a single 2.4 kb transcript abundant in Torpedo electric organ , moderately expressed in spinal cord and electric lobe, and undetecta ble in non-neural tissues. Immunohistochemistry shows that NSIST is ex pressed in a punctate distribution in the innervated portion of electr ocytes. In the CNS, NSIST-like immunoreactivity is localized within th e somas of motor neurons and neurons of the electromotor nucleus, wher eas mAb-3B3 immunostaining is associated with cell surfaces and neurop il. Neuronal NSIST is therefore likely to exert its effects extracellu larly; although NSIST is synthesized by neurons, its product, the 3B3 epitope, is found outside neuronal cell bodies. Our evidence indicates that NSIST participates in nervous system specific posttranslational modifications, perhaps including those at synapses.