Identification, isolation, and characterization of CNS glycoproteins that bear N-linked oligosaccharides terminating in beta 1,4-linked GalNAc-4-SO4

Glycoprotein oligosaccharides and carbohydrate specific receptors play a significant role in mammalian biological function. The extent of these biological functions is often reflected in the temporal and spatial regulation of glycosylation of target proteins as well as the repertoire of carbohydrate specific receptors in the vicinity. Our lab has shown that GalNAc-4-SO₄ on the circulating lutropin (LH) binds to the Man/GalNAc-4-SO₄-receptor from liver which mediates its rapid clearance from the blood, a function that is essential for generating the episodic rise and fall in circulatory LH levels required for stimulation of ovulation and implantation.; Synthesis of oligosaccharides with terminal GalNAc-4-SO₄ requires coordinate expression a protein specific GalNAc-transferase and the GalNAc-4-sulfotransferase within a cell or tissue. Besides the pituitary, only a few tissues express both enzyme activities, including the cerebellum, hippocampus, and cerebral cortex from the brain, suggesting that these areas of the brain express glycoproteins bearing terminal N-linked GalNAc-4-SO₄.; Tenascin-R (TN-R) is a multifunctional extracellular matrix component exclusively expressed in the central nervous system (CNS). The expression of TN-R by oligodendrocytes and interneurons in the hippocampus, cerebellum, and cerebral cortex is highly regulated during development. We determined that TN-R associated with cerebellar Purkinje cells and their dendrites is the major glycoprotein bearing N-linked oligosaccharides terminating in GalNAc-4-SO ₄ in the cerebellum. Further, staining with the GalNAc-4-SO₄ specific, Cys-Fc chimera in hippocampus and cerebral cortex significantly overlapped with known expression patterns for TN-R, suggesting that TN-R is the major target for GalNAc-4-SO₄ modification in the CNS. The neural cell line SHSY5Y expresses TN-R, both GalNAc and sulfotransferases, and a small number of glycoproteins that bear oligosaccharides with N-linked terminal GalNac-4-SO₄. Taken together with the overlapping localizations of TN-R, GalNAc-4-SO₄, and small interneurons in cerebellum, hippocampus and cerebral cortex, we conclude that neuronally expressed TN-R is modified with N-linked oligosaccharides terminating in GalNAc-4-SO ₄. Finally, the Man/GalNAc-4-SO₄-receptor is expressed throughout development on the surface of perivascular microglia in the CNS, and interaction with GalNAc-4-SO₄ on TN-R with this and other GalNAc-4-SO ₄ specific receptors in the CNS may mediate the function of TN-R in neuronal growth, stabilization, and synaptogenesis.