Lianwen Zhang

Overexpression and topology of bacterial oligosaccharyltransferase PglB.

Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N(cyt)-C(peri) topology with 11 transmembrane segments for the STT3 family proteins.

A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase.

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) is widely distributed on nucleocytoplasmic proteins and participates in various physiological processes. But O‐GlcNAc status on numerous proteins remains unknown. To better understand this modification, computational analysis combined with experimental study was performed in this work. Structural analysis of many O‐GlcNAcylation sites indicated that the modification occurred predominantly in a random coil region. Frequency analysis on many O‐GlcNAcylated peptides revealed a signature sequence, PPVS/TSATT, around the modification site (underlined, position 0). Based on the sequence, a peptide panel was designed to investigate key positions affecting O‐GlcNAcylation of peptides and their amino acid preference. It was indicated that 3 positions (–2, –1, and +2) had an important role for this modification, where the presence of uncharged amino acids with small side chains could confer high reactivity. The amino acid preference at key positions was further investigated on bovine crystalline αvia site‐directed mutagenesis. The preferred amino acids were Pro > Ala > Gly at position – 2, Ala > Thr > Val > Lys > Pro at position –1, and Ala > Gly > Arg > Glu at position +2. Altogether, these findings suggested that a substrate (peptide or protein) with Pro, Ala at position –2, and/or Val, Ala, Thr, Ser at position –1, and/or Ala, Ser, Pro, Thr, Gly at position +2 would have more chances for O‐GlcNAcylation. To test the rule, 2 O‐GlcNAcylation sites on sOGT (S52 and T449) were predicted and confirmed by Western blot. The present work systematically investigated the sequence signature for O‐GlcNAcylation. The result will contribute to predicting the O‐GlcNAc status of a protein and further functional studies.—Liu, X., Li, L., Wang, Y., Yan, H., Ma, X., Wang, P. G., Zhang, L. A peptide panel investigation reveals the acceptor specificity of O‐GlcNAc transferase. FASEB J. 28, 3362–3372 (2014). www.fasebj.org

Synthesis of flavonol 3-O-glycoside by UGT78D1

Glycosylation is an important method for the structural modification of various flavonols, resulting in the glycosides with increased solubility, stability and bioavailability compared with the corresponding aglycone. From the physiological point of view, glycosylation of plant flavonoids is of importance and interest. However, it is notoriously complicated that flavonols such as quercetin, kaempferol and myricetin, are glucosylated regioselectively at the specific position by chemical method. Compared to the chemical method, enzymatic synthesis present several advantages, such as mild reaction condition, high stereo or region selectivity, no protection/deprotection and high yield. UGT78D1 is a flavonol-specific glycosyltransferase, responsible for transferring rhamnose or glucose to the 3-OH position in vitro. In this study, the activity of UGT78D1 was tested against 28 flavonoids acceptors using UDP-glucose as donor nucleoside in vitro, and 5 acceptors, quercetin, myricetin, kaempferol, fisetin and isorhamnetin, were discovered to be glucosylated at 3-OH position. Herein, the small-scale 3-O-glucosylated quercetin, kaempferol and myricetin were synthesized by UGT78D1 and their chemical structures were confirmed by 1H and 13C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS).

Substrate Specificity Provides Insights into the Sugar Donor Recognition Mechanism of O-GlcNAc Transferase (OGT)

O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism.

An easy colorimetric assay for glycosyltransferases

Glycosyltransferases are involved in biosynthesis of both protein-bound and non-bound glycans that have multiple and important biological functions in all species. A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. In this work, a novel colorimetric assay for the glycosyltransferase-catalyzed reaction was established. Compared with measuring the newly formed product, which might not exhibit visible absorption, the unreacted acceptor could be readily detected by measuring the visible absorption of the hydrolysis product. In the assay, 4-nitrophenyl-β-D-glycoside (glycosyl-β-pNP) is used as the glycosyl acceptor, which can be hydrolyzed by a special exoglycosidase to release the p-nitrophenol before glycosylation reactions. Absorbance change of the p-nitrophenolate corresponds to unreacted glycosyl acceptor that accompanied the glycosyl transfer. The assay is demonstrated to be useful in the initial characterization of recombinant glycosyltransferases for their kinetic parameters, optimal metal cofactor, and pH value. It provides a simple, sensitive, and quantitative method for assessing glycosyltransferase activity and is thus expected to have broad applications including automated high-throughput screening.

Isoforms of human O-GlcNAcase show distinct catalytic efficiencies

O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63–342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the Km value and catalytic efficiency (kcat/Km) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest Km value of 0.26 mM and the highest catalytic efficiency of 3.51·103. These results reveal that the N-terminal region (a.a. 1–350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.

Proteomic analysis of O-GlcNAcylated proteins in invasive ductal breast carcinomas with and without lymph node metastasis

The potential role of protein O-GlcNAcylation in cancer has been studied extensively, and the spread of cancer cells to regional lymph nodes is the first step in the dissemination of breast cancer. However, the correlation between O-GlcNAcylation and lymphatic metastasis in breast cancer remains elusive. In this study, we demonstrated that the overall O-GlcNAcylation as well as O-GlcNAc transferase (OGT) tends to decrease in response to the augmentation of lymph node metastasis (LNM) in invasive ductal breast carcinomas (IDCs). Although accumulating evidence indicates that individual O-GlcNAcylation may be important in the pathogenesis of breast cancer, O-GlcNAcylated proteins in IDCs are still largely unexplored. Herein, O-GlcNAcylated proteins of IDCs were chemo-enzymatically enriched and identified via liquid chromatography combined with tandem mass spectrometry. In total, 155 O-GlcNAcylated proteins were determined, of which 41 were only observed in LNM tissues, while 40 were unique in non-LNM samples. Gene ontology analysis showed that O-GlcNAc is primarily a nucleocytoplasmic post-translational modification, and most enriched functional terms were related to cancer development in both metastatic and non-metastatic IDCs. Moreover, several O-GlcNAcylated proteins involved in glycolysis and its accessory pathway were identified from LNM and non-LNM groups, respectively. These results indicate that the O-GlcNAcylation statuses of individual proteins were independent of the overall O-GlcNAcylation levels of metastatic and non-metastatic IDCs. Aberrant O-GlcNAc modification of these proteins might be associated with LNM progression.

Site-Directed Glycosylation of Peptide/Protein with Homogeneous O-Linked Eukaryotic N-Glycans.

Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.

Structural and biochemical insights into nucleotide-rhamnose synthase/epimerase-reductase from Arabidopsis thaliana.

L-Rhamnose (Rha) is synthesized via a similar enzymatic pathway in bacteria, plants and fungi. In plants, nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER) catalyzes the final step in the conversion of dTDP/UDP-α-D-Glc to dTDP/UDP-β-L-Rha in an NAD(P)H dependent manner. Currently, only biochemical evidence for the function of NRS/ER has been described. In this study, a crystal structure for Arabidopsis thaliana NRS/ER was determined, which is the first report of a eukaryotic rhamnose synthase with both epimerase and reductase activities. NRS/ER functions as a metal ion independent homodimer that forms through hydrophobic interactions via a four-helix bundle. Each monomer exhibits α/β folding that can be divided into two regions, nucleotide cofactor binding domain and sugar substrate binding domain. The affinities of ligands with NRS/ER were measured using isothermal titration calorimetry, which showed that NRS/ER has a preference for dTDP over UDP, while the cofactor binding site has a similar affinity for NADH and NADPH. Structural analysis coupled to site-directed mutagenesis suggested C115 and K183 as the acid/base pair responsible for epimerization, while T113, Y144 and K148 are the conserved residues in reduction. These findings shed light on the molecular mechanism of NRS/ER and were helpful to explore other eukaryotic enzymes involved in L-Rha synthesis.

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation.