Showing papers by "Yoshiki Yamaguchi published in 2015"

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

Abstract: The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics.

Backbone 1H, 13C, and 15N resonance assignments of the Fc fragment of human immunoglobulin G glycoprotein

Abstract: The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions. Here, we report NMR assignments of the glycosylated Fc fragment (Mr 53 kDa), cleaved from a chimeric antibody with human IgG1 constant regions, which was produced in Chinese hamster ovary cells with uniform 13C- and 15N-labeling. Electronic supplementary material The online version of this article (doi:10.1007/s12104-014-9586-7) contains supplementary material, which is available to authorized users.

Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides

Abstract: ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical-shift perturbation experiments with uniformly (15)N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p.

Identification of matrix metalloproteinase inhibitors by chemical arrays.

Abstract: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand–protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest ...

In situ visualization of a glycoform of transferrin: localization of α2,6-sialylated transferrin in the liver.

Abstract: We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to α2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing α2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.

Glycan Marker for Idiopathic Normal Pressure Hydrocephalus

Abstract: Two glycan isoforms of transferrin (Tf), Tf-1 and Tf-2, were found in cerebrospinal fluid (CSF). Tf-1 concentration in CSF was reduced in idiopathic normal pressure hydrocephalus (iNPH), an elderly dementia caused by abnormal metabolism of CSF, whereas Tf-2 concentration was not. The reduction of Tf-1 concentration was not found in Alzheimer’s disease, indicating that Tf-1 can distinguish iNPH from Alzheimer’s disease. Glycan analysis revealed that Tf-1 has unique biantennary asialoand agalacto-complex N-glycans, which carry bisecting b1,4-GlcNAc and core a1,6-fucose. In contrast, Tf-2 has a2,6-sialylglycan like serum Tf, suggesting that Tf-2 is derived from serum. Tf-1, a glycoform unique to CSF, is speculated to be produced in brain tissue. Immunohistochemistry using anti-Tf antibody on brain tissues revealed that the choroid plexus, which produces CSF, was strongly stained by anti-Tf antibody. These results suggest that Tf-1 is secreted from the choroid plexus and its secretion is reduced by abnormal metabolism of CSF in iNPH.