No abstract

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

Background: High expression levels of core fucosylated N-glycans in brain tissues remain unexplained. Results: Loss of core fucosylation enhanced AMPA receptor heteromerization and decreased long term potentiation. Conclusion: Core fucosylation is required for hippocampal long term potentiation. Significance: Core fucosylation may be very important for the neuronal synaptic plasticity that is required for learning and memory.

The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.