Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b

The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex₃HexNAc₄Fuc₁ glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man₈GlcNAc₂ glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.     

ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis

N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different alpha-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LLO.     

Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I)

Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc₃Man₉GlcNAc₂-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man₅GlcNAc₂-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man₅GlcNAc₂-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man₅GlcNAc₂-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labeling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients’ cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn²⁺ for optimal activity. In CDG patients’ cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.     

Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [³H]mannose from the [³H]Man₉GlcNAc as well as for releasing mannose from p-nitrophenyl-α-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca²⁺ was the most effective cation for reversing this inhibition. Mn²⁺ was considerably less effective than Ca²⁺ and Mg²⁺ was without effect. The processing mannosidase was inhibited by α1,2- and α1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and α1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [³H]Man₉GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man₅GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific α1,2-mannosidase.     

Glycoengineering of host mimicking type‐2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces

Bacterial carbohydrate structures play a central role in mediating a variety of host–pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram‐negative bacteria, is composed of a lipid A‐core and the O‐antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1–4(Fucα1–3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram‐negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O‐antigen. A ubiquitous building block in mammalian N‐linked protein glycans is Galβ1‐4GlcNAc, referred to as a type‐2 N‐acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A‐cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly‐LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI‐TOF and NMR analysis. Glycoengineered LOS induced pro‐inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.     

BIOCHEMICAL CHARACTERIZATION OF A NOVEL β‐N‐ACETYLHEXOSAMINIDASE FROM THE INSECT OSTRINIA FURNACALIS

The β‐N‐acetylhexosaminidase FDL specifically removes the β‐1,2‐GlcNAc residue conjugated to the α‐1,3‐mannose residue of the core structure of insect N‐glycans, playing significant physiological roles in post‐translational modification in the Golgi apparatus. Little is known about its enzymatic properties. We obtained the OfFDL gene from the insect Ostrinia furnacalis by RT‐PCR. The full length cDNA of FDL is 2241 bp carrying an opening reading frame of 1923 bp encoding 640 amino acids. The recombinant protein OfFDL in a soluble and active form was obtained with high purity through a two‐step purification strategy. The recombinant OfFDL exclusively hydrolyzes the terminal β‐1,2‐GlcNAc residue from the α‐1,3 branch instead of the α‐1,6 branch of the substrate GnGn‐PA. Several kinetic parameters including k_cat/K_m values toward four artificial substrates and K_i values of three representative hexosaminidase inhibitors were obtained.     

Crystal structure of a fully glycosylated HIV‐1 gp120 core reveals a stabilizing role for the glycan at Asn262

The crystal structure of a fully glycosylated HIV‐1 gp120 core in complex with CD4 receptor and Fab 17b at 4.5‐Å resolution reveals 9 of the 15 N‐linked glycans of core gp120 to be partially ordered. The glycan at position Asn262 had the most extensive and well‐ordered electron density, and a GlcNAc₂Man₇ was modeled. The GlcNAc stem of this glycan is largely buried in a cleft in gp120, suggesting a role in gp120 folding and stability. Its arms interact with the stems of neighboring glycans from the oligomannose patch, which is a major target for broadly neutralizing antibodies. Proteins 2015; 83:590–596. © 2014 Wiley Periodicals, Inc.     

Mannose 6‐phosphate‐independent Lysosomal Sorting of LIMP‐2

The lysosomal integral membrane protein type 2 (LIMP‐2/SCARB2) has been described as a mannose 6‐phosphate (M6P)‐independent trafficking receptor for β‐glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP‐2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP‐2 with the cation‐independent M6P receptor (MPR) results in M6P‐dependent targeting of LIMP‐2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P‐dependent delivery of LIMP‐2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP‐2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P‐forming N‐acetylglucosamine (GlcNAc)‐1‐phosphotransferase, LIMP‐2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP‐2 levels within lysosomes purified from liver of wild‐type (wt) and GlcNAc‐1‐phosphotransferase‐defective mice. Heterologous expression of the luminal domain of LIMP‐2 in wild‐type, LIMP‐2‐deficient and GlcNAc‐1‐phosphotransferase‐defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP‐2. Finally, cathepsin Z, a known GlcNAc‐1‐phosphotransferase substrate, but not LIMP‐2, could be precipitated with M6P‐specific antibodies. These data prove M6P‐independent lysosomal sorting of LIMP‐2 and subsequently GC in vivo.      

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg²⁺‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl₂, but became disrupted in the absence of MgCl₂, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.     

Lack of the evidence for the enzymatic catabolism of Man1GlcNAc2 in Saccharomyces cerevisiae

In the cytosol of Saccharomyces cerevisiae, most of the free N-glycans (FNGs) are generated from misfolded glycoproteins by the action of the cytoplasmic peptide: N-glycanase (Png1). A cytosol/vacuole α-mannosidase, Ams1, then trims the FNGs to eventually form a trisaccharide composed of Manβ1,4GlcNAc β1,4GlcNAc (Man₁GlcNAc₂). Whether or not the resulting Man₁GlcNAc₂ is enzymatically degraded further, however, is currently unknown. The objective of this study was to unveil the fate of Man₁GlcNAc₂ in S. cerevisiae. Quantitative analyses of the FNGs revealed a steady increase in the amount of Man₁GlcNAc₂ produced in the post-diauxic and stationary phases, suggesting that this trisaccharide is not catabolized during this period. Inoculation of the stationary phase cells into fresh medium resulted in a reduction in the levels of Man₁GlcNAc₂. However, this reduction was caused by its dilution due to cell division in the fresh medium. Our results thus indicate that Man₁GlcNAc₂ is not enzymatically catabolized in S. cerevisiae.     

Identification of high-mannose and multiantennary complex-type <Emphasis Type="Italic">N</Emphasis>-linked glycans containing α-galactose epitopes from Nurse shark IgM heavy chain

MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man₆GlcNAc₂ accompanied by small amounts of Man₅GlcNAc₂, Man₇GlcNAc₂ and Man₈GlcNAc₂. Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (β1→4-linked to the central mannose) and with varying numbers of α-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.     

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

Crystal structures of Gα_i (and closely related family member Gα_t) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Gα_t exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP‐bound state, unlike Gα_i, which exhibits higher basal exchange and a disordered Switch II region in Gα_iGDP structures. Using purified Gα_i and Gα_t, we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Gα proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GαGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation‐π interaction between tryptophan and arginine residues in the Switch II of Gα_i family proteins that mediates the observed red shift in emission maxima. Furthermore, amino‐terminal myristoylation of Gα_i resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Gα proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.