Molecular cloning of cDNAs encoding α1, α2, and β subunits of rat brain platelet-activating factor acetylhydrolase1The nucleotide sequence data reported in this paper have been submitted to the GenBank database under the accession number AF016047 for α1, AF016048 for α2 and AF016049 for β, respectively.1

AbstractBrain intracellular platelet-activating factor acetylhydrolase (PAF-AH(Ib)) is a tertiary G-protein-complex-like heterotrimeric enzyme which is composed of α1, α2, and β subunits and is implicated in stages of brain development such as the formation of the brain cortex. We have isolated and sequenced cDNA clones encoding these three subunits of rat brain PAF-AH(Ib). The amino acid sequences of brain PAF-AH has shown an extremely high homology among mammalian species. The tissue distribution of the three subunits was examined by Northern blot analysis. Although the mRNAs were detected in various organs, the ratio of the level of mRNA expression for the three subunits differed among rat tissues, raising the possibility that isoform(s) other than the heterotrimeric isoform exist in certain tissues

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer\u27s disease

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\u27s 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

Major glycan structure underlying expression of the Lewis X epitope in the developing brain is O-mannose-linked glycans on phosphacan/RPTPβ.

Glycosylation is a major protein modification. Although proteins are glycosylated/further modulated by several glycosyltransferases during trafficking from the endoplasmic reticulum to the Golgi apparatus, a certain glycan epitope has only been detected on a limited number of proteins. Of these glycan epitopes, Lewis X is highly expressed in the early stage of a developing brain and plays important roles in cell-cell interaction. The Lewis X epitope is comprised of a trisaccharide (Galβ1-4 (Fucα1-3) GlcNAc), and a key enzyme for the expression of this epitope is α1, 3-fucosyltransferase 9. However, the scaffolding glycan structure responsible for the formation of the Lewis X epitope as well as its major carrier protein has not been fully characterized in the nervous system. Here we showed that the Lewis X epitope was mainly expressed on phosphacan/receptor protein tyrosine phosphatase β (RPTPβ) in the developing mouse brain. Expression of the Lewis X epitope was markedly reduced in β1, 4-galactosyltransferase 2 (β4GalT2) gene-deficient mice, which indicated that β4GalT2 is a major galactosyltransferase required for the Lewis X epitope. We also showed that the Lewis X epitope almost disappeared due to the knockout of protein O-mannose β1, 2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for the synthesis of O-mannosylated glycans, which indicated that the O-mannosylated glycan is responsible for presenting the Lewis X epitope. Since O-mannosylated glycans on phosphacan/RPTPβ could also present human natural killer-1, another glycan epitope specifically expressed in the nervous system, our results revealed the importance of O-mannosylated glycan chains in the presentation of functional glycan epitopes in the brain

Toolbox Accelerating Glycomics (TAG): Improving Large-Scale Serum Glycomics and Refinement to Identify SALSA-Modified and Rare Glycans

Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N’-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses

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 structure of POMGNT2 provides new insights into the mechanism to determine the functional O‐mannosylation site on αα‐dystroglycan

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds β1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds β1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure