Expression Cloning and Characterization of NSIST, a Novel Sulfotransferase Expressed by a Subset of Neurons and Postsynaptic Targets

Abstract

Synapses are distinguished by localized concentrations of specific proteins, many of which bear the marks of posttranslational processing such as glycosylation and sulfation. One strategy to elucidate this posttranslational tailoring is to identify the enzymes that create these modifications. Monoclonal antibody 3B3 recognizes a carbohydrate-containing epitope expressed on dystroglycan and other constituents of Torpedo electric organ synaptic membranes. We used mAb 3B3 in an immunofluorescence-based expression-cloning method and isolated a cDNA clone conferring mAb-3B3 immunoreactivity to transfected COS cells. The deduced polypeptide has a predicted molecular weight of 51 kDa, a type II transmembrane topology, and four potential N-linked glycosylation sites. The polypeptide, which we term NSIST (nervous system involved sulfotransferase), shows extensive, although not complete, homology to a chondroitin-6-sulfotransferase and limited homology to other sulfotransferases. In NSIST-transfected COS cells, 35SO4 incorporation and chondroitin-sulfate-like immunoreactivity are increased. In vivo, NSIST occurs as a single 2.4 kb transcript abundant in Torpedo electric organ, moderately expressed in spinal cord and electric lobe, and undetectable in non-neural tissues. Immunohistochemistry shows that NSIST is expressed in a punctate distribution in the innervated portion of electrocytes. In the CNS, NSIST-like immunoreactivity is localized within the somas of motor neurons and neurons of the electromotor nucleus, whereas mAb-3B3 immunostaining is associated with cell surfaces and neuropil. Neuronal NSIST is therefore likely to exert its effects extracellularly; 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.