Probing enzymatic properties of N-glycosyltransferase isoforms from Mannheimia haemolytica.

N-Glycosyltransferase (NGT) is an inverting glycosyltransferase for an unusual pathway of N-linked protein glycosylation and glycosylates polypeptides in the consensus sequon (N-(X≠P)-T/S) with hexose monosaccharides. Here, we expressed and characterized a novel N-glycosyltransferase from Mannheimia haemolytica (named MhNGT). RP-HPLC and Mass Spectrometry were used to assay and quantify glycopeptide formation by MhNGT and determine its substrate specificities. MhNGT could utilize a variety of nucleotide-activated sugar donors, including UDP-Glc, UDP-Gal and UDP-Xyl, to glycosylate the tested peptides DANYTK, GGNWTT and PAVGNCSSALR with higher efficiency than ApNGT which was comprehensive studied. The optimum temperature of MhNGT was about 30 °C and the optimum pH was 7.5-8.0 in PBS-NaOH buffer. MhNGT exhibited a different position-specific residue preference of substrate peptides from the NGT of Actinobacillus pleuropneumoniae (ApNGT). The effective glycosylation of common short peptides by MhNGT demonstrated the enzyme's potential to alter therapeutically significant mammalian N-glycoproteins.

O-GalNAc glycosylation affects the immunogenicity of the receptor-binding domain (RBD) of SARS-CoV-2 spike protein.

The spike protein of SARS-CoV-2 has been widely used as an effective vaccine immunogen, although some limitations still remain. Herein, O-GalNAc glycosylated RBD (Tn-RBD) was synthesized as an antigen via in vitro glycosylation reactions. The inhibition ability against hACE2 binding of antibodies induced with Tn-RBD was 30-40% increased compared to that induced with RBD. This result implies that Tn-glycosylation might play important roles in the immunogenicity of the RBD protein, which should be considered in the design of novel vaccines to fight against COVID-19.

Chemoenzymatic Synthesis of SARS-CoV-2 Homogeneous O-Linked Glycopeptides for Exploring Their Inhibition Functions.

Harnessing highly conserved peptides derived from the receptor binding domain (RBD) of spike (S) protein to construct peptide-based inhibitors is one of the most effective strategies to fight against the ever-mutating coronavirus SARS-CoV-2. But how the O-glycosylation affects their inhibition abilities has not been intensively explored. Herein, an intrinsic O-glycosylated peptide P320-334 derived from RBD was screened and homogeneous O-linked glycopeptides containing Tn (GalNAcα1-O-Ser/Thr), T (Galß1-3GalNAcα1-O-Ser/Thr), sialyl-Tn (sTn, Siaα2-6GalNAcα1-O-Ser/Thr), and sialyl-T (sT, Siaα2-3Galß1-3GalNAcα1-O-Ser/Thr) structures were first synthesized via chemoenzymatic strategies. Compared with the unglycosylated peptide, the binding of sT-P320-334 to hACE2 was enhanced to 133% and the inhibition capacity against RBD-hACE2 binding of sTn- and sT-P320-334 was significantly increased up to 150-410%. Thus, our results suggest the sialic acid residue on the terminal of short O-glycan structures might strengthen the inhibition capacities of these peptide-based inhibitors, which might provide novel optimization directions for the inhibitor design.

Substrate specificity of the galactokinase from the human gut symbiont Akkermansia muciniphila ATCC BAA-835.

Galactokinases, which catalyze the phosphorylation of galactose and possible other monosaccharides, can provide an activated sugar donor to synthesize sugar-containing molecules. In this study, a novel galactokinase from human gut symbiont Akkermansia muciniphila ATCC BAA-835 (GalKAmu) was expressed and characterized. GalKAmu displayed broad substrate tolerance, with catalytic activity towards Gal (100 %), GalN (100 %), GalA (20.2 %), Glc (52.5 %), GlcNAc (15.5 %), Xyl (<5%), ManNAc (58 %), ManF (37.4 %) and l-Glc (80 %). Most interestingly, this was the first GalK isoform which can tolerate ManNAc. Thus, our characterization of GalKAmu broadens the substrate selection of galactokinases.

Arabidopsis REM16 acts as a B3 domain transcription factor to promote flowering time via directly binding to the promoters of SOC1 and FT.

Actin depolymerizing factor (ADF) is a key modulator for dynamic organization of actin cytoskeleton. Interestingly, it was found that the ADF1 gene silencing delays flowering, but its mechanism remains unclear. In this study, ADF1 was used as a bait to screen its interacting proteins by the yeast two-hybrid (Y2H) system. One of them, the REM16 transcription factor was identified. As one of the AP2/B3-like transcriptional factor family members, the REM16 contains two B3 domains and its transcript levels kept increasing during the floral transition stage. Overexpression of REM16 accelerates flowering while silencing of REM16 delays flowering. Gene expression analysis indicated that the key flowering activation genes such as CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) were upregulated in the REM16 overexpression lines, while the transcription of the flowering suppression gene FLOWERING LOCUS C (FLC) was decreased. In contrast, the REM16 gene silencing lines contained lower transcript levels of the CO, FT, LFY and SOC1 but higher transcript levels of the FLC compared with the wild-type plants. It was proved that REM16 could directly bind to the promoter regions of SOC1 and FT by in vitro and in vivo assays. Genetic analysis supported that REM16 acts upstream of SOC1 and FT in flowering pathways. All these studies provided strong evidence demonstrating that REM16 promotes flowering by directly activating SOC1 and FT.

Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris.

BACKGROUND: Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. RESULTS: In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. CONCLUSION: This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.

Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species.

N-glycosyltransferase (NGT) is responsible for transferring hexose monosaccharides to the asparagine side chain of proteins and polypeptides in the consensus sequon (N-(X≠P)-T/S) with nucleotide-activated sugars as donor substrates. Here, we expressed and purified four different N-glycosyltransferases derived from diverse bacteria, including Actinobacillus pleuropneumoniae, Aggregatibacter aphrophilus, Kingella kingae and Bibersteinia trehalosi, and measured their catalytic activities of four synthesized peptides via in vitro glycosylation assays. RP-HPLC and mass spectrometry were used to identify and quantify the glycopeptide formation by distinct NGT isoforms. We then analyzed and compared the glycosylation efficiencies of different peptides for these four NGT isoforms, which showed distinct substrate selectivities. We sought to probe peptide specificities among various NGT isoforms, which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.

Expression, purification and characterization of ROP66-178 GTPase from Arabidopsis thaliana.

The unique type of GTPases in plants, termed ROPs, are the small GTP-binding proteins involved in signal transduction which play important roles in regulation of hormonal response pathway, cell polarity, defense from plant pathogens, etc. In order to explore the regulation mechanism of AtROPs involved in, the purified ROPs were needed to explore the interactions of ROP GTPases with their regulators and effectors. In this study, the first ROP GTPase from Arabidopsis thaliana, AtROP66-178 was successfully expressed in Escherichia coli and obtained in high quality and purity through affinity chromatography and gel-filtration chromatography. The resultant protein was identified as a single band of 19 kDa in SDS-PAGE and was confirmed to be active to interact with guanine nucleotides through the fluorescence-based assay. The intrinsic tryptophan fluorescence intensity of AtROP66-178 was enhanced upon interacting with either GDP or GTP. Meanwhile, the equilibrium dissociation constants of AtROP66-178 with fluorescent guanine nucleotide analogue mantGDP and mantGTP were determined to be 0.0721 µM and 0.0422 µM, respectively, based on fluorescence polarization.