A sulfated glycosaminoglycan linkage region is a novel type of human natural killer-1 (HNK-1) epitope expressed on aggrecan in perineuronal nets

Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity.We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockoutmice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutantmice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockoutmice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and-S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity

Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains.

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

The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3. IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression

HNK-1 carbohydrate and aggrecan are expressed in the PNNs.

<p>Sagittal sections of cerebral cortex from 6-week-old WT (A-C) and PKO mice (D-L) were singly (for WT) or doubly (for PKO) immunostained with HNK-1 mAb (A, D), 6B4 mAb (B, G), or Cat-315 mAb (C, J), and aggrecan pAb (E, H, K). F, I and L are overlaid images. High magnification images of HNK-1-, 6B4- or Cat-315- and aggrecan-positive PNNs are shown in the insets. Scale bars, 200 μm (A-C), 100 μm (D-L) and 20 μm (insets).</p

Biosynthetic model for HNK-1 epitope on aggrecan in PNNs.

<p>GlcAT-I is responsible for synthesis of a linkage region of GAG, which is usually further elongated into a long GAG chain (e.g., CS chain). HNK-1ST transfers a sulfate group to GlcA of the linkage region of aggrecan, which likely results in the expression of the linkage type HNK-1 epitope, HSO₃-GlcA-Gal-Gal-Xyl, in PNNs. The expression of the HNK-1 epitope on aggrecan suppresses the CS polymerization that starts from GlcA of the linkage region.</p

HNK-1 carbohydrate epitope expressed in GlcAT-P-deficient mice.

<p>(A, B) Soluble fractions prepared from 2- and 6-week-old mouse brains (WT and GlcAT-P-deficient mice) were treated with chABC with or without PNGase F, subjected to SDS-PAGE and then blotted using anti-HNK-1 mAb, 6B4 mAb, Cat-315 mAb, phosphacan pAb and aggrecan pAb. To compare the molecular weight of HNK-1 immunoreactive band with aggrecan or phosphacan, an HNK-1 mAb blot of 6-week-old PKO mice treated with chABC and PNGase F is shown on the right of the aggrecan panel. (C) Using urea-soluble fractions from 6-week-old PKO mouse brains (input), aggrecan was immunoprecipitated using aggrecan pAb (IP: aggrecan) or normal rabbit IgG (IP: IgG). The precipitated aggrecan was subjected to SDS-PAGE and western blotting with HNK-1, 6B4, Cat-315 and aggrecan antibodies.</p

LC/MSⁿ structural analysis of HNK-1 epitope on aggrecan.

<p>(A) PA-labeled <i>O</i>-linked glycans were prepared from aggrecan-Fc co-expressed with HNK-1ST in COS-1 cells. The base peak chromatogram of the glycans was obtained using selected ion monitoring (SIM) (<i>m/z</i> 782.5–832.5) in the negative ion mode (<i>upper panel</i>). An extracted ion chromatogram (EIC) of the ion at <i>m/z</i> 807.0–807.4 (<i>lower panel</i>). (B) MS/MS spectra (<i>upper panel</i>) of the ion [M-H]^- (<i>m/z</i> 807.2) detected in peak A and MS/MS/MS spectra (<i>lower panel</i>) of the predominant product ion (<i>m/z</i> 727.2) in the MS/MS. S, sulfate group; HexA, hexuronic acid; Hex, hexose; Xyl-PA, xylose labeled with 2-aminopyridine.</p

HNK-1 carbohydrate expressed in PKO and DKO mice.

<p>(A) Soluble fractions from 6-week-old GlcAT-P-deficient mice (PKO) and GlcAT-P and -S double deficient mice (DKO) were treated with chABC and PNGase F, subjected to SDS-PAGE and blotted using HNK-1 mAb, 6B4 mAb, Cat-315 mAb, aggrecan pAb, phosphacan pAb, H28 (NCAM mAb) or actin mAb. The effect of PNGase F treatment was confirmed by western blotting with H28 mAb. Actin was used as the loading control. (B) Using soluble fractions from 6-week-old DKO mouse brains (input), aggrecan and phosphacan were immunoprecipitated with aggrecan pAb (IP: aggrecan), normal rabbit IgG (IP: IgG) or phosphacan pAb (IP: phosphacan). The precipitated aggrecan and phosphacan were subjected to SDS-PAGE and western blotting with HNK-1, aggrecan and phosphacan antibodies. (C-N) Sagittal sections of cerebral cortex from 6-week-old PKO (C-E) and DKO mice (F-N) were double-immunostained with HNK-1 mAb, 6B4 mAb or Cat-315 mAb (<i>red</i>) and WFA lectin (<i>green</i>). Scale bar, 100 μm.</p