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, 35 SO 4 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.

The Small Leucine-Rich Repeat Proteoglycan Biglycan Binds to α-Dystroglycan and Is Upregulated in Dystrophic Muscle

“…It is possible that synapse-rich tissues contain specific enzymes that modify the chondroitin sulfate side chains. Interestingly, at least one such enzyme, the chondroitin-6-sulfotransferase NSIST, is selectively enriched in Torpedo electric organ and brain (Nastuk et al, 1998). Together, these results suggest that the interaction between dystro-glycan and biglycan may be highly regulated through posttranslational modification.…”

Epitope Diversity of N-Glycans from Bovine Peripheral Myelin Glycoprotein P0 Revealed by Mass Spectrometry and Nano Probe Magic Angle Spinning 1H NMR Spectroscopy

“…In this context, the presence of the 6-Osulfo-GlcNAc oligosaccharides (fractions Q1.4 -2.10) and specifically the 6-O-sulfo-HNK-1 epitope, observed in fraction Q2.10 is interesting since it implies another sulfotransferase activity. Of the various 6-O-sulfo-GlcNAc-transferases identified thus far (49 -52, 79), only one report describes the existence of a nervous system-involved sulfotransferase (53), which showed a high degree of homology to a family of 6-O-sulfotransferases, some of which are expressed also in brain (50,54,80,81).…”

Small Molecule Inhibitors of the Sulfotransferases

“…For instance, sulfation of carbohydrate epitopes on activated endothelial cells alerts circulating leukocytes to sites of chronic inflammation [13][14][15][16]. In addition, sulfation of cell surface and secreted glycoproteins mediates growth factor binding [17], regulates blood clotting [18][19][20] and cell differentiation [21,22], and elicits astrocyte and neuron outgrowth [23,24]. Unlike the cytosolic subfamily, however, the Golgi STs have been difficult to characterize at the biochemical level.…”