Streamlined Subclass-Specific Absolute Quantification of Serum IgG Glycopeptides Using Synthetic Isotope-Labeled Standards.

Absolute glycoproteomics quantification has drawn tremendous attention owing to its prospects in biomarker discovery and clinical implementation but is impeded by a general lack of suitable heavy isotope-labeled glycopeptide standards. In this study, we devised a facile chemoenzymatic strategy to synthesize a total of 36 human IgG glycopeptides attached with well-defined glycoforms, including 15 isotope-labeled ones with a mass increment of 6 Da to their native counterparts. Spiking of these standards into human sera enabled simplified, robust, and precise absolute quantification of IgG glycopeptides in a subclass-specific fashion. Additionally, the implementation of the absolute quantification approach revealed subclass-dependent alteration of serum IgG galactosylation and sialylation in colon cancer samples.

Penicillium oxalicum putative methyltransferase Mtr23B has similarities and differences with LaeA in regulating conidium development and glycoside hydrolase gene expression.

Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene-melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.

Three glycoside hydrolase family 12 enzymes display diversity in substrate specificities and synergistic action between each other.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze ß-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-ß-glucanase; it preferentially hydrolyzes mix-linked ß-glucan (barley ß-glucan, ß-1,3-1,4-glucan) and slightly hydrolyzes ß-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward ß-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

Enzymatic rhamnosylation of anticancer drugs by an α-L-rhamnosidase from Alternaria sp. L1 for cancer-targeting and enzyme-activated prodrug therapy.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Rational designed mutagenesis of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity study.

Levansucrases, which belong to the glycoside hydrolase family 68 (GH68), synthesize ß (2-6)-linked fructan levan with sucrose as substrate. We described the use of a levansucrase (Bl_SacB) from Bacillus licheniformis 8-37-0-1 for catalysis of fructosyl transfer to obtain high levan yield previously. In the present study, six variants (Y246A, N251A, K372A, R369A, R369S, and R369K) were constructed through sequence alignment and structural analysis to explore the synthesis mechanism of Bl_SacB. The selected residues were predicted to localize to the substrate-entering channel of the active cavity and close to or remote from the catalytic triad. The products of these variants ranged from homopolymers levan to fructo-oligosaccharides (FOSs). The primary FOSs were identified through MS and NMR analyses as neolevan-type neokestose [ß-D-Fru-(2-6)-α-D-Glc-(1-2)-ß-D-Fru], levan-type 6-kestose [ß-D-Fru-(2-6)-ß-D-Fru-(2-1)-α-D-Glc], and inulin-type 1-kestose [ß-D-Fru-(2-1)-ß-D-Fru-(2-1)-α-D-Glc]. The mutation at Tyr246 located remote from the catalytic triad led to the production of short-chain oligosaccharides with degree of polymerization (DP) of up to 25. The replaced Arg369 located close to the catalytic triad resulted in either elimination of polysaccharide synthesis or complete change in the dominant linkage of the products. The Michaelis constants (Km) of Y246A, N251A, K372A, and R369K were found to be similar to that of the wild type (WT). However, the turnover number (kcat) and the value of transfructosylation versus hydrolysis activity of the six variants decreased compared with those of the WT. Hence, the residues located on the surface of the substrate-entering channel of Bl_SacB can be critical in product linkage type and/or elongation mechanism.

Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.

N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H218O. Subsequent LC-MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification. Graphical abstract Proposed mechanism of glycopeptides enrichment using DEAE-Sepharose.

Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.

Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galß1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Convenient and Precise Strategy for Mapping N-Glycosylation Sites Using Microwave-Assisted Acid Hydrolysis and Characteristic Ions Recognition.

N-glycosylation is one of the most prevalence protein post-translational modifications (PTM) which is involved in several biological processes. Alternation of N-glycosylation is associated with cellular malfunction and development of disease. Thus, investigation of protein N-glycosylation is crucial for diagnosis and treatment of disease. Currently, deglycosylation with peptide N-glycosidase F is the most commonly used technique in N-glycosylation analysis. Additionally, a common error in N-glycosylation site identification, resulting from protein chemical deamidation, has largely been ignored. In this study, we developed a convenient and precise approach for mapping N-glycosylation sites utilizing with optimized TFA hydrolysis, ZIC-HILIC enrichment, and characteristic ions of N-acetylglucosamine (GlcNAc) from higher-energy collisional dissociation (HCD) fragmentation. Using this method, we identified a total of 257 N-glycosylation sites and 144 N-glycoproteins from healthy human serum. Compared to deglycosylation with endoglycosidase, this strategy is more convenient and efficient for large scale N-glycosylation sites identification and provides an important alternative approach for the study of N-glycoprotein function.

Chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose and study of the substrate specificity of HldE.

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.

The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.

Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis.

Two novel synthetic α2-6-linked disialyl hexasaccharides, disialyllacto-N-neotetraose (DSLNnT) and α2-6-linked disialyllacto-N-tetraose (DS'LNT), were readily obtained by highly efficient one-pot multienzyme (OPME) reactions. The sequential OPME systems described herein allowed the use of an inexpensive disaccharide and simple monosaccharides to synthesize the desired complex oligosaccharides with high efficiency and selectivity. DSLNnT and DS'LNT were shown to protect neonatal rats from necrotizing enterocolitis (NEC) and are good therapeutic candidates for preclinical experiments and clinical application in treating NEC in preterm infants.

One-pot multi-enzyme (OPME) chemoenzymatic synthesis of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural or non-natural sialic acid.

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5'-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5'-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni ß1-3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2-3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.

Temperature-dependent mycotoxins production investigation in Alternaria infected cherry by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry.

For temperature-dependent Alternaria mycotoxins production analysis, cherry samples were inoculated with Alternaria sp. and incubated at two different temperatures (4 °C and 25 °C). Six Alternaria mycotoxins, including altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), altertoxin-I (ATX-I), tenuazonic acid (TeA), and tentoxin (TEN), in cherries were detected with integrated visible data-processing tools. Maximum concentration of these mycotoxins reached 71,862.2 µg/kg at 25 °C. Notably, considerable amount of TeA (290.4 µg/kg) was detected at 4 °C, which indicated that low temperature is not a safe storage condition for fruits. A total of 102 compounds were detected with a neutral loss of 162.0528 Da, and TeA-glucose was identified in this work. Based on MS/MS cosine similarity, products were verified and annotated with feature based molecular networking (FBMN) in global natural products social networking (GNPS). The results showed Alternaria mycotoxins in cherry samples were mainly demethylation, hydrogenation, and dehydration. This work revealed the production of Alternaria mycotoxins in cherries under different storage temperature, which will provide theoretical basis for the control of mycotoxin contamination in food commodities.

Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2.

Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

[Expression of a SARS-CoV-2 neutralizing nanobody in Trichoderma reesei].

Nanobodies derived from camelid single-chain antibodies have the advantages of being small, simple, highly soluble and stable. Nanobodies can be administered by inhalation and therefore is potentially valuable for the prevention and control of respiratory viruses. Trichoderma reesei is a food-grade protein expression host with a cellulase production capacity of up to 80 g/L, which can be employed for low-cost production of therapeutic proteins. In this study, a codon-optimized SARS-CoV-2 neutralizing nanobody Nb20 was expressed in T. reesei under a strong constitutive promoter Pcdna1. Nb20 protein was fused downstream of the N-terminal fragment of cellobiohydrolase Ⅰ, and the fusion protein can be intracellularly cleaved by the KEX2 protease to release Nb20. In a shake-flask fermentation using glucose medium, 47.4 mg/L Nb20 was detected in the culture after 48 h of cultivation. The expressed Nb20 showed the ability to interact with the receptor-binding domain of SARS-CoV-2 spike protein, suggesting that it can be used for the neutralization of SARS-CoV-2. The results indicate that T. reesei has the potential for recombinant production of nanobodies.

Substrate promiscuity of N-acetylhexosamine 1-kinases.

N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180-185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

Comparative glycosylation mapping of plasma-derived and recombinant human factor VIII.

Human coagulation factor VIII (FVIII) is a key co-factor in the clotting cascade, the deficiency of which leads to Hemophilia A. Human plasma-derived (pdFVIII) and recombinant FVIII (rFVIII) had been used as effective products to prevent and treat bleeding episodes. Both FVIII products share identical amino acid sequences and appear to be equivalent as of clinical efficiency. However, systemic reviews found an increased risk of neutralizing antibody (or inhibitor) development with recombinant products. FVIII is a highly glycosylated protein, and its glycosylation pattern is specific to host cells and environments. The roles of glycosylation in immune responses toward pdFVIII and rFVIII are yet to be defined. Herein, we systemically profiled N- and O-glycomes of pdFVIII and rFVIII using a mass spectrometry-based glycoproteomic strategy. A total of 110 site-specific N-glycopeptides consisting of 61 N-glycoforms were identified quantitatively from rFVIII and pdFVIII. Additionally, 31 O-glycoforms were identified on 23 peptides from rFVIII and pdFVIII. A comprehensive comparison of their site-specific glycan profiles revealed distinct differences between the glycosylation of pdFVIII and rFVIII.

Glucose oxidase and polydopamine functionalized iron oxide nanoparticles: combination of the photothermal effect and reactive oxygen species generation for dual-modality selective cancer therapy.

Cancer cells possess some inherent characteristics, such as glucose-dependence and intolerance to heat and exogenous reactive oxygen species (ROS). In this study, a strategy has been developed to target these vulnerable weaknesses of cancer cells using glucose oxidase (GOx) and polydopamine (PDA) functionalized iron oxide nanoparticles (Fe3O4@PDA/GOx NPs). PDA is first deposited on the surfaces of iron oxide NPs through self-polymerization, and then GOx is covalently linked with PDA upon mixing the enzyme and Fe3O4@PDA under alkaline conditions. In this system, the PDA layer along with iron oxide NPs serves as a photothermal transfer material converting near infrared (NIR) radiation into heat. The covalently linked GOx can competitively consume glucose and spontaneously generate ROS H2O2 that can be further converted by the iron oxide NPs into more toxic ˙OH, inducing apoptosis of cancer cells. The selective toxicity of Fe3O4@PDA/GOx NPs on cancer cells is demonstrated both in vitro and in vivo. In particular, a single injection rather than multiple doses results in significant suppression of tumors, and does not induce apparent histological lesions in the 4T1 tumor-bearing Balb/c mice. The versatility of the functionalization strategy reported in this study will contribute to developing efficient therapies for selective cancer treatment.

Identification of Key Components for the Optimization of Cellulase Mixtures Using a Proteomic Strategy.

Efficient degradation of complex lignocellulosic materials requires the synergistic action of different types of enzymes. Characterizing the compositions of lignocellulolytic enzyme mixtures could provide comprehensive understandings about the enzymatic degradation of lignocelluloses. In this chapter, we present a proteomic strategy for the analysis of enzyme mixtures produced by lignocellulolytic fungi. The described method is easy to carry out and is suitable to determine the composition of lignocellulolytic enzyme mixtures in a semiquantitative manner. Comparison of the compositions of enzyme mixtures with different degrading efficiencies allows for the identification of candidate targets for the optimization of lignocellulolytic enzyme mixtures.

A general strategy for the synthesis of homogeneous hyaluronan conjugates and their biological applications.

Here, we developed a general strategy for synthesizing homogeneous HA conjugates, and generated homogeneous HA-pNP, HA-biotin, and HA-oroxylin conjugates to investigate the relationships between HA chain length and its diverse biological functions.