Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

Identification and characterization of a novel thermo-stable endo-ß-N-acetylglucosaminidase from Rhizomucorpusillus.

Endo-ß-N-acetylglucosaminidase (ENGase) is an enzyme that hydrolyzes the chitobiose core of N-glycans and is widely used for glycan analysis on glycoproteins and preparation of precursors for glycosylated compounds. While most of the ENGases that can hydrolyze complex-type glycans are derived from eukaryotes, their production by heterologous expression using Escherichia coli is insufficient, making the production process expensive. From an industrial perspective, there is a need for a less expensive enzyme with higher activity and stability. In this study, we identified a novel ENGase gene from a thermophilic fungus, Rhizomucor pusillus, and named it Endo-Rp. Characterization of the recombinant Endo-Rp showed that the enzyme had maximum hydrolytic activity at 60 °C and hydrolyzed high-mannose-type and biantennary complex-type glycans, but not (2,4)-branched triantennary complex-type or fucosylated glycans. Endo-Rp also hydrolyzed N-glycans attached to RNase B and human transferrin. In summary, we consider Endo-Rp to be a valuable enzyme in various scientific and industrial applications.

Transcriptome-wide association study reveals two genes that influence mismatch negativity.

Mismatch negativity (MMN) is a differential electrophysiological response measuring cortical adaptability to unpredictable stimuli. MMN is consistently attenuated in patients with psychosis. However, the genetics of MMN are uncharted, limiting the validation of MMN as a psychosis endophenotype. Here, we perform a transcriptome-wide association study of 728 individuals, which reveals 2 genes (FAM89A and ENGASE) whose expression in cortical tissues is associated with MMN. Enrichment analyses of neurodevelopmental expression signatures show that genes associated with MMN tend to be overexpressed in the frontal cortex during prenatal development but are significantly downregulated in adulthood. Endophenotype ranking value calculations comparing MMN and three other candidate psychosis endophenotypes (lateral ventricular volume and two auditory-verbal learning measures) find MMN to be considerably superior. These results yield promising insights into sensory processing in the cortex and endorse the notion of MMN as a psychosis endophenotype.

[Cloning, expression and characterization of a new endo-ß-N-acetylglucosaminidase from Streptomyces alfalfae].

Endo-ß-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-ß-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×106 U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis.

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants.

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.

Bacteriophage T4 capsid as a nanocarrier for Peptide-N-Glycosidase F immobilization through self-assembly.

Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and laborious immobilization procedures has limited its industrial application and commercialization. In this study, we report a novel self-assembly immobilization system using bacteriophage T4 capsid as a nanocarrier. The system utilizes the binding sites of the small outer capsid protein, Soc, on the T4 capsid. Enzymes as Soc fusions constructed with regular molecular cloning technology expressed at the appropriate time during phage assembly and self-assembled onto the capsids. The proof of principle experiment was carried out by immobilizing ß-galactosidase, and the system was successfully applied to the immobilization of an important glycomics enzyme, Peptide-N-Glycosidase F. Production of Peptide-N-Glycosidase F and simultaneous immobilization was finished within seven hours. Characterizations of the immobilized Peptide-N-Glycosidase F indicated high retention of activity and well reserved deglycosylation capacity. The immobilized Peptide-N-Glycosidase F was easily recycled by centrifugation and exhibited good stability that sustained five repeated uses. This novel system uses the self-amplified T4 capsid as the nanoparticle-type of supporting material, and operates with a self-assembly procedure, making it a simple and low-cost enzyme immobilization technology with promising application potentials.

Selective deglycosylation of lactoferrin to understand glycans' contribution to antimicrobial activity of lactoferrin.

Lactoferrin is a highly glycosylated antimicrobial protein that contains multiple glycan types. In this research, recombinantly produced three forms of novel endo-ß-N-acetylglucosaminidase (free, genetically attached Glutatiohine-S-transferase and polyhistide) were used for selective release of lactoferrin glycans to understand the contribution of specific glycan types to the antimicrobial function of lactoferrin. Three lactoferrin forms with different glycan profile were obtained by treatment with these fusion tagged enzymes; native, fully deglycosylated and sialylated glycan enriched lactoferrin. The released glycan structures were analyzed and confirmed with mass spectrometry. The results showed that native and sialylated glycans enriched lactoferrin have similar minimum inhibitory concentration (MIC) values against E.coli DH5a (1 mg/ml), whereas the MIC value for fully deglycosylated lactoferrin was 6mg/ml. These results suggest that sialylated glycans play important role in the antimicrobial function of lactoferrin.

Characterization of novel endo-ß-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG.

Endo-ß-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3-2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.

Structural modeling and mutagenesis of endo-ß-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Endo-ß-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-ß-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-ß-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-ß-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-ß-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Synthesis of a hybrid type N-glycan heptasaccharide oxazoline for Endo M catalysed glycosylation.

Endo-ß-N-acetylglucosaminidases (ENGases) are versatile biocatalysts that allow access to a wide variety of defined homogenous N-linked glycoconjugates in a convergent manner. A hybrid-type N-glycan was accessed by total synthesis, converted to an oxazoline, and used as a donor substrate with both wild type Endo M and an N175Q glycosynthase Endo M mutant allowing the convergent synthesis of a glycosylated amino acid bearing a hybrid N-glycan structure.

Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum.

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.

Glycan conversion of glycoprotein via the transglycosylation activity of endo-ß-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.

Kinetic characterization of a novel endo-ß-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

EndoBI-1 is a recently isolated endo-ß-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.

A cholesterol consensus motif is required for efficient intracellular transport and raft association of a group 2 HA from influenza virus.

The HA (haemagglutinin) of influenza viruses must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. We previously showed using FRET that deletion of the two raft-targeting features of HA, S-acylation at the cytoplasmic tail and the hydrophobic amino acids VIL (Val-Ile-Leu) in the outer part of the TMR (transmembrane region), lead to reduced raft association. In addition, exchange of VIL, but not of the S-acylation sites severely retards transport of HA through the Golgi. In the present study, we have further characterized the ill-defined signal in the TMR. A sequence comparison suggests that the leucine residue of VIL might be part of a CCM (cholesterol consensus motif) that is known to bind cholesterol to seven-transmembrane receptors. The signal also comprises a lysine residue and a tryptophan residue on one and a tyrosine residue on another TMR helix and is conserved in group 2 HAs. Mutations in the CCM retard Golgi-localized processing of HA, such as acquisition of Endo H (endoglycosidase H)-resistant carbohydrates in the medial Golgi and proteolytic cleavage in the TGN (trans-Golgi network). The delay in transport of HA to and from the medial Golgi varied with the mutation, suggesting that different transport steps are affected. All mutants analysed by FRET also showed reduced association with rafts at the plasma membrane. Thus the raft-targeting signal of HA encompasses not only hydrophobic, but also aromatic and positively charged, residues. We speculate that binding to cholesterol might facilitate intracellular transport of HA and association with rafts.

EndoE from Enterococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth.

Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-ß-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.

Expression, purification, and characterization of endo-ß-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system.

Endo-ß-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.