Effects Of O-glycosylation Research Articles

Effect of O-glycosylation on amyloid fibril formation of the variable domain in the Vλ6 light chain mutant Wil

Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation.

Nuclear Localized O-Fucosyltransferase SPY Facilitates PRR5 Proteolysis to Fine-Tune the Pace of Arabidopsis Circadian Clock

Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

A study on enhanced O-glycosylation strategy for improved production of recombinant human chorionic gonadotropin in Chinese hamster ovary cells

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and β subunit linked with disulfide bridges. The β subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the β subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC–MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.

Substituent Effects on in Vitro Antioxidizing Properties, Stability, and Solubility in Flavonoids

Antioxidants are widely used by humans, both as dietary supplements and as additives to different types of products. The desired properties of an antioxidant often include a balance between the antioxidizing capacity, stability, and solubility. This review focuses on flavonoids, which are naturally occurring antioxidants, and different common substituent groups on flavonoids and how these affect the properties of the molecules in vitro. Hydroxyl groups on flavonoids are both important for the antioxidizing capacity and key points for further modification resulting in O-methylation, -glycosylation, -sulfation, or -acylation. The effects of O-glycosylation and acylation are discussed as these types of substitutions have been most explored in vitro concerning antioxidizing properties as well as stability and solubility. Possibilities to control the properties by enzymatic acylation and glycosylation are also reviewed, showing that depending on the choice of enzyme and substrate, regioselective results can be obtained, introducing possibilities for more targeted production of antioxidants with predesigned properties.

Effect of O-glycosylation and tyrosinesulfation of leech-derived peptides on binding and inhibitory activity against thrombin

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.

An Efficient Approach for the Characterization of Mucin‐Type Glycopeptides: The Effect of O‐Glycosylation on the Conformation of Synthetic Mucin Peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.

The Stabilizing Effects of O-glycosylation on the Secondary Structural Integrity of the Designed α-loop-α motif by Molecular Dynamics Simulations

In this study, various 400 ps molecular dynamics simulations were conducted to determine the stabilizing effect of O-glycosylation on the secondary structural integrity of the design α-loop-α motif, which has the optimal loop length of 7 Gly residues (denoted as N-A16G7A16-C). In general, O-glycosylation stabilizes the structural integrity of the model peptide regardless of the length and position of glycosylation sites because it decreases the opportunity for water molecules to compete for the intramolecular hydrogen bonds. The designed peptide exhibits the highest helicity when residues 11 and 31 are replaced with Ser residues followed by O-linked with 3 galactose residues, representing the “face-to-face” glycosylation near the loop. In this case, the loop exhibits an extended conformation and several new hydrogen bonds are observed between the main chain of the loop and the galactose residues, resulting in decreasing the fluctuation and increasing the stability of the entire peptide. When the glycosylation are made close to the loop, the secondary structural integrity of the α-loop-α motif increases with the number of galactose residues. In addition, “face- to-face” glycosylation increases the structural integrity of this motif to a greater extent than “back-to-back” glycosylation. However, when the glycosylation are created away from the loop and near the N- and C-termini, no general rule is found for the stabilizing effect.

Molecular Dynamics Simulations of the O-glycosylated 21-residue MUC1 Peptides

The conformational propensities of the 21-residue peptide and its Oglycosylated analogs were studied by molecular dynamics (MD) simulations. This polypeptide motif comprises the tandem repeat of the human mucin (MUC1) protein core that is differently glycosylated in normal and cancer cells. To evaluate the structural effects of O-glycosylation on the polypeptide backbone, conformations of the nonglycosylated peptide and its glycosylated analogs were monitored during the 1 ns MD simulations. Radius gyration for whole peptide and its fragments, as well as root-meansquare-deviation between coordinate sets of the backbone atoms of starting structures and generated structures, were calculated. It was shown that O-glycosylation promotes and stabilizes the extended conformations of the whole peptide and its central PDTRP fragment. O-glycosylation of the specific Thr residues significantly affects the conformational distributions of the flanking Ser residues. It was also shown that Oglycosylation promoted backbone conformations of the immunodominant region PDTRP that were similar to the structural features of the peptides presented by the major histocompatability complex (MHC) to T-cell receptors.

O-glycosylation delays the clearance of human IGF-binding protein-6 from the circulation.

The actions of insulin-like growth factors (IGF-I and IGF-II) are modulated by a family of six structurally related, high-affinity binding proteins (IGFBPs 1-6). IGFBP-6, an O-linked glycoprotein, preferentially binds IGF-II and inhibits its actions. The aim of this study was to investigate whether O-glycosylation modulates the pharmacokinetics of IGFBP-6. The pharmacokinetic profiles of (125)I-labelled glycosylated (g) and non-glycosylated (n-g) recombinant human IGFBP-6 were studied following intravenous bolus administration in anaesthetised rats. The redistribution half-life of gIGFBP-6 was 2.3-fold greater than that of n-gIGFBP-6 (14.4+/- 1.2 vs 6.3+/-1.5 min, P=0. 006). The elimination half-life of gIGFBP-6 was 21-fold greater than that of n-gIGFBP-6 (584.2+/-130.2 vs 28.0+/-4.2 min, P=0.019). The effect of O-glycosylation on IGFBP-6 pharmacokinetics was not due to inhibition of intravascular proteolysis. Radioactivity was found in stomach, kidneys, lung, spleen, heart and liver but not brain 4h after injection of g or n-gIGFBP-6. O-glycosylation delays the clearance of IGFBP-6 from the circulation and may therefore contribute to its role as a circulating inhibitor of IGF-II actions.

Glycosylations versus conformational preferences of cancer associated mucin core.

Synthetic oligosaccharide vaccines based on core STn (sialyl alpha2-6 GalNAc) carbohydrate epitopes are being evaluated by a number of biopharmaceutical firms as potential immunotherapeutics in the treatment of mucin-expressing adenocarcinomas. The STn carbohydrate epitopes exist as discontinuous clusters, O-linked to proximal serine and threonine residues within the mucin sequence. In an effort to probe the structure and dynamics of STn carbohydrate clusters as they may exist on the cancer-associated mucin, we have used NMR spectroscopy and MD simulations to study the effect of O-glycosylation of adjacent serine residues in a repeating (Ser)n sequence. Three model peptides/glyco-peptides were studied: a serine trimer containing no carbohydrate groups ((Ser)3 trimer); a serine trimer containing three Tn (GalNAc) carbohydrates alpha-linked to the hydroxyls of adjacent serine sidechains ((Ser.Tn)3 trimer); and a serine trimer containing three STn carbohydrates alpha-linked to the hydroxyls of adjacent serine sidechains ((Ser.STn)3 trimer). Our results demonstrate that clustering of carbohydrates shifts the conformational equilibrium of the underlying peptide backbone into a more extended and rigid state, an arrangement that could function to optimally present the clustered carbohydrate antigen to the immune system. Steric effects appear to drive these changes since an increase in the size of the attached carbohydrate (STn versus Tn) is accompanied by a stronger shift in the equilibrium toward the extended state. In addition, NMR evidence points to the formation of hydrogen bonds between the peptide backbone NH protons and the proximal GalNAc groups in the (Ser.Tn)3 and (Ser.STn)3 trimers. The putative peptide-sugar hydrogen bonds may also play a role in influencing the conformation of the underlying peptide backbone, as well as the orientation of the O-linked carbohydrate. The significance of these results will be discussed within the framework of developing clustered STn-based vaccines, capable of targeting the clustered STn epitopes on the cancer-associated mucin.

NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate-peptide interactions.

Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galbeta (1-3)GalNAc] O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by 1H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I) and APPETTAAPPT (series II) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and nonaqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galbeta (1-3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly L-proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.

Crystal structure of a fully protected β- O-galactosylated tripeptide

The crystal structure of the fully protected glycotripeptide N-benzyloxycarbonyl- O-(2,3,4,6-tetra- O-acetyl-β- d-galactopyranosyl)- l-threonyl-α-aminoisobutyryl-α-aminoisobutyric acid tert-butyl ester [Z-(β- d-GalAc 4)- l-Thr-Aib-Aib-O tBu] has been determined by X-ray diffraction. The peptide backbone is fully extended at Thr(1), left-handed helical at Aib(2), while it is right-handed helical at Aib(3). The fully extended conformation at the N-terminal Thr residue is stabilized by an intramolecular H-bond involving the N-1–H and O-1C-1 groups (intramolecularly H-bonded C 5 conformation). Two additional intramolecular H-bonds are observed, involving the peptide N–H groups of Aib(2) and Aib(3) as the donors, and the pyranosidic O-5 oxygen atom and the carbonyl oxygen atom of the acetoxy group on C-6 of the sugar, respectively, as the acceptors. Owing to the peptide–sugar H-bonds, the peptide backbone is forced to adopt a conformation dramatically different from the β-bend/3 10-helical conformation usually observed for Aib-rich peptides. The implications and limitations of these findings on the effect of O-glycosylation on the conformation of natural peptides are briefly outlined.

Recognition of Peptidyl Epitopes by Polymorphic Epithelial Mucin (PEM)-Specific Monoclonal Antibodies

Peptidyl epitope recognition by several murine monoclonal antibodies (MAbs E29, H23, HMFG-1, HMFG-2, MA5, MA6 and MA9) which react with the polymorphic epithelial mucins [PEM; epithelial membrane antigen (EMA)] was studied by using ten synthetic peptides representative of the 20 residue tandem repeat as test antigens. Antibody binding to 6-10 residue overlaps and to peptides having a common carboxy-terminus and staggered amino-termini (8-31 residues) was assessed by solid phase and competition ELISA techniques. From these analyses, all MAbs except MA9 were found to react predominantly with the carboxy-terminal half of the repeat motif. Polyclonal antibody responses in mice immunized with intact EMA/PEM-containing preparations also displayed significant reactivities against synthetic repeat peptide antigens and, conversely, synthetic peptides as carrier-conjugated immunogens induced antibodies recognizing intact antigens. These results are discussed vis-à-vis peptide conformation, the potential effects of O-glycosylation on secondary structure, and the possible effects of these parameters on immunogenicity and antigenicity.

Expression of ApoE Gene in Chinese Hamster Cells with a Reversible Defect in O-Glycosylation: Glycosylation is Not Required for ApoE Secretion

The effects of O-glycosylation on the synthesis and secretion of apolipoprotein E (apoE, a glycoprotein with O- but not N-linked sugars) were studied with a UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase-deficient cell mutant (ldlD cells) which expresses a reversible defect in protein O-glycosylation. Under normal culture conditions the mutant ldlD cells cannot add N-acetylgalactosamine (GalNAc) to proteins. GalNAc is the first sugar of mucin-type O-linked oligosaccharides attached to the protein. This O-glycosylation defect is rapidly corrected when GalNAc is added to the culture medium. These cells also require external sources of galactose for the addition of this sugar to O-linked and other oligosaccharides. A bovine papilloma virus-based expression vector for human apoE and the human metallothionein 1A gene were transfected into ldlD cells, and apoE-expressing cell clones resistant to CdCl2 were selected and used in the present studies. The structure and secretion of apoE in these cells were examined by immunoprecipitation and one- and two-dimensional gel electrophoresis and autoradiography. The synthesis, rate, and extent of secretion of apoE were unaffected by O-glycosylation (GalNAc-independent). In the presence of both galactose and GalNAc, multiple apoE isoforms were synthesized in ldlD cells as a result of variation in the extent of sialylation. ApoE sialylation was dependent on the addition of galactose as well as GalNAc to the extracellular medium, suggesting that addition of galactose to the nascent oligosaccharide chains was required for the addition of sialic acid.