Tissue-specific regulation of hnk-1 biosynthesis by bisecting glcnac

Human natural killer—1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc

神経回路形成期におけるホスファカンには特徴的なO-マンノース型HNK-1糖鎖が発現する

京都大学0048新制・課程博士博士(人間健康科学)甲第18198号人健博第15号新制||人健||2(附属図書館)31056京都大学大学院医学研究科人間健康科学系専攻(主査)教授 齋藤 邦明, 教授 三谷 章, 教授 髙橋 良輔学位規則第4条第1項該当Doctor of Human Health SciencesKyoto UniversityDFA

Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

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

Site-specific HNK-1 epitope on alternatively spliced fibronectin type-III repeats in tenascin-C promotes neurite outgrowth of hippocampal neurons through contactin-1

The human natural killer-1 (HNK-1) carbohydrate epitope, composed of a unique sulfated trisaccharide (HSO3–3GlcAβ1–3Galβ1–4GlcNAc-R), is highly expressed during brain development and regulates higher brain function. However, it remains unclear which glycoprotein carries the HNK-1 epitope in the embryonic brain and the functional role it plays. Here, we showed that one of the major HNK-1 carrier proteins in the embryonic brain is tenascin-C (TNC), an extracellular matrix protein that regulates neurite outgrowth by interacting with the GPI-anchored protein contactin-1 (CNTN). Because the alternatively spliced fibronectin type-III (FNIII) repeats in TNC give rise to many isoforms and affect neuronal function, we evaluated neurite outgrowth of primary hippocampal neurons on purified recombinant FNIII repeats with or without the HNK-1 epitope as a substrate. We found that the presence of the HNK-1 epitope on the C domain of TNC promoted neurite outgrowth, and that this signal was mediated by CNTN, which is an HNK-1-expressing neuronal receptor. The neurite-promoting activity of the HNK-1 epitope on TNC required neuronal HNK-1 expression, which was defective in neurons lacking the glucuronyltransferases GlcAT-P and GlcAT-S. These results suggest that the HNK-1 epitope is a key modifier of TNC and CNTN in the regulation of embryonic brain development

Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1–4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits’ ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate

The HNK-1 epitope on N-glycan at N413 enhances cell surface expression of GluA2.

<p>(A) A cell biotinylation assay was applied to HEK293 cells expressing GluA2 (WT, N256S, or N413S) with (+) or without (-) HNK-1-synthesizing enzymes (GlcAT-P and HNK-1ST). Biotinylated GluA2 was immunoblotted with anti-GluA2/3 polyclonal antibody (Surface). The cell lysates were also immunoblotted with the same polyclonal antibody (Total) and an HNK-1 monoclonal antibody. (B) Relative intensities of surface expression levels of GluA2 (surface/total) were calculated and normalized with that of WT without the HNK-1 epitope.</p

The HNK-1 epitope expressed on GluA2 enhances cell surface expression of GluA1.

<p>A cell biotinylation assay was applied to HEK293 cells expressing GluA1 with (+) or without (-) GluA2 or HNK-1-synthesizing enzymes (GlcAT-P and HNK-1ST) as indicated. Biotinylated GluA1 and GluA2 were immunoblotted with anti-GluA1 and anti-GluA2/3 polyclonal antibodies, respectively (Surface) (A). The cell lysates were also immunoblotted with the same polyclonal antibodies (Total) and an HNK-1 monoclonal antibody (B). (C) Relative intensities of surface expression levels of GluA2 or GluA1 (surface/total) were calculated and normalized with or without the HNK-1 epitope.</p

AMPA receptors in the synapse turnover by monomer diffusion

記憶の鍵となる受容体を働かせる仕組みを解明 --4つのパーツを0.1秒毎に組み立て直して働かせるメカニズム--. 京都大学プレスリリース. 2019-11-26.The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1–4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition. However, by observing GluA1 and GluA2 at the level of single molecules, we find that the homo- and heterotetramers are metastable, instantaneously falling apart into monomers, dimers, or trimers (in 100 and 200 ms, respectively), which readily form tetramers again. In the dendritic plasma membrane, GluA1 and GluA2 monomers and dimers are far more mobile than tetramers and enter and exit from the synaptic regions. We conclude that AMPAR turnover by lateral diffusion, essential for sustaining synaptic function, is largely done by monomers of AMPAR subunits, rather than preformed tetramers