Carving out a Glycoside Hydrolase Active Site for Incorporation into a New Protein Scaffold Using Deep Network Hallucination.

Enzymes are indispensable biocatalysts for numerous industrial applications, yet stability, selectivity, and restricted substrate recognition present limitations for their use. Despite the importance of enzyme engineering in overcoming these limitations, success is often challenged by the intricate architecture of enzymes derived from natural sources. Recent advances in computational methods have enabled the de novo design of simplified scaffolds with specific functional sites. Such scaffolds may be advantageous as platforms for enzyme engineering. Here, we present a strategy for the de novo design of a simplified scaffold of an endo-α-N-acetylgalactosaminidase active site, a glycoside hydrolase from the GH101 enzyme family. Using a combination of trRosetta hallucination, iterative cycles of deep-learning-based structure prediction, and ProteinMPNN sequence design, we designed proteins with 290 amino acids incorporating the active site while reducing the molecular weight by over 100 kDa compared to the initial endo-α-N-acetylgalactosaminidase. Of 11 tested designs, six were expressed as soluble monomers, displaying similar or increased thermostabilities compared to the natural enzyme. Despite lacking detectable enzymatic activity, the experimentally determined crystal structures of a representative design closely matched the design with a root-mean-square deviation of 1.0 Å, with most catalytically important side chains within 2.0 Å. The results highlight the potential of scaffold hallucination in designing proteins that may serve as a foundation for subsequent enzyme engineering.

Potent and Selective Cell-Active Iminosugar Inhibitors of Human α-N-Acetylgalactosaminidase (α-NAGAL).

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N-acetyl-α-galactosaminidase (α-NAGAL), the GH responsible for cleaving terminal α-N-acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non-carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α-NAGAL selective (∼200-fold) guanidino-containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image-based method to measure levels of the Tn-antigen, a cellular glycoprotein substrate of α-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α-NAGAL within cells using patient derived fibroblasts (EC50 =150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal ß-hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off-target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α-NAGAL.

Low α-N-acetylgalactosaminidase plasma concentration correlates with the presence and severity of the bipolar affective disorder.

OBJECTIVES: Believing that a neurodevelopmental pathology may cause bipolar affective disorder (BAD), we aimed to measure the concentrations of α-N-acetylgalactosaminidase (α-NAGAL), a lysosomal enzyme. METHODS: The study included 32 patients with BAD and 32 healthy controls. The Young Mania Rating Scale was used to measure the severity of the disease. Serum α-N-acetylgalactosaminidase concentrations were measured in all blood samples using the human α-N-acetylgalactosaminidase ELISA Kit. RESULTS: A statistically significant difference was found in the α-NAGAL values between the groups. The mean α-NAGAL values of BAD patients are lower than the mean α-NAGAL values of the control group. A strong negative and statistically significant relationship was found between the α-NAGAL values of patients with BAD and their Young Mania Rating Scale scores. And a positive strong correlation was found between the age of onset of the disease and α-NAGAL levels. CONCLUSIONS: Low α-N-acetylgalactosaminidase concentrations may cause the accumulation of some glycoproteins in the lysosomes in the brain during the gestational period, producing the clinical symptoms of BAD. α-N-acetylgalactosaminidase deficiency may not be the only cause of BAD, but it may be an important factor in the aetiology of this disease.

Could low α-N-acetylgalactosaminidase plasma concentration cause schizophrenia?

OBJECTIVES: Using a neurodevelopmental approach to examine the aetiology, we predicted an enzyme deficiency to exist at the cellular level and aimed to measure α-N-acetylgalactosaminidase (α-NAGAL) blood levels. METHODS: The study included 32 patients diagnosed with schizophrenia and 30 healthy controls. The positive and negative syndrome scale (PANSS) was applied to the patients with schizophrenia. Serum α-NAGAL concentrations were measured in blood samples taken from all participants using the human alpha-N-acetylgalactosaminidase ELISA Kit. RESULTS: The mean α-NAGAL values of schizophrenic patients are lower than the mean α-NAGAL values of the control group (p = 0.000 < 0.001). Correlation analysis showed that there was a significant relationship between α-NAGAL values and PANSS scores of patients with schizophrenia. PANSS total (r = -0.708, p = 0.000 < 0.001), PANSS positive (r = -0.627, p = 0.000 < 0.001), PANSS negative (r = -0.386, p = 0.029 < 0.05). And a positive moderate correlation was found between the age of onset of the disease and α-NAGAL levels (r = 0.529, p = 0.002 < 0.05). CONCLUSIONS: Based on the neurodevelopmental hypothesis, the low α-NAGAL concentrations this study found might cause accumulation of glycoproteins in the lysosomes in the central nervous system during the gestational period and then might result in the clinical symptoms of schizophrenia. α-NAGAL may be an important factor in the aetiology of schizophrenia.

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galß1-3GalNAc reactivity on Fetuin.

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galß1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galß1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galß1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galß1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galß1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galß1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.

Immunotherapy with GcMAF revisited - A critical overview of the research of Nobuto Yamamoto.

This overview describes the research of Nobutu Yamamoto (Philadelphia) concerning immunotherapy with GcMAF for patients with cancer and for patients infected with pathogenic envelope viruses. GcMAF (Group-specific component Macrophage-Activating Factor) is a mammalian protein with an incredible potency to directly activate macrophages. Since the late 1980s Yamamoto's investigations were published in numerous journals but in order to understand the details of his research, a minute survey of many of his patents was required. But even then, regrettably, a precise description of his experiments was sometimes lacking. This overview tries to summarize all of Yamamoto's research on GcMAF, as well as some selected more recent papers from other investigators, who tried to verify and/or reproduce Yamamoto's reports. In my opinion the most important result of the GcMAF research deserves widespread renewed attention: human GcMAF injections (100 ng per week, intramuscular or intravenous) can help to cure patients with a great variety of cancers as well as patients infected with pathogenic envelope viruses like the human immunodeficiency virus 1 (HIV-1), influenza, measles and rubella (and maybe also SARS-CoV-2). From Yamamoto's data it can be calculated that GcMAF is a near-stoichiometric activator of macrophages. Yamamoto monitored the progress of his immunotherapy via the serum level of an enzyme called nagalase (α-N-acetylgalactosaminidase activity at pH 6). I have extensively discussed the properties and potential catalytic site of this enzyme activity in an Appendix entitled: "Search for the potential active site of the latent α-N-acetylgalactosaminidase activity in the glycoproteins of some envelope viruses".

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Identification of recurrent pathogenic alleles using exome sequencing data: Proof-of-concept study of Russian subjects.

Whole exome sequencing (WES) is a powerful tool for the cataloguing of population-specific genetic diseases. Within this proof-of-concept study we evaluated whether analysis of a small number of individual exomes is capable of identifying recurrent pathogenic alleles. We considered 106 exomes of subjects of Russian origin and revealed 13 genetic variants, which occurred more than twice and fulfilled the criteria for pathogenicity. All these alleles turned out to be indeed recurrent, as revealed by the analysis of 1045 healthy Russian donors. Eight of these variants (NAGA c.973G>A, ACADM c.985A>C, MPO c.2031-2A>C, SLC3A1 c.1400T>C, LRP2 c.6160G>A, BCHE c.293A>G, MPO c.752T>C, FCN3 c.349delC) are non-Russian-specific, as their high prevalence was previously demonstrated in other European populations. The remaining five disease-associated alleles appear to be characteristic for subjects of Russian origin and include CLCN1 c.2680C>T (myotonia congenita), DHCR7 c.453G>A (Smith-Lemli-Opitz syndrome), NUP93 c.1162C>T (steroid-resistant nephrotic syndrome, type 12), SLC26A2 c.1957T>A (multiple epiphyseal dysplasia) and EIF3F c.694T>G (mental retardation). These recessive disease conditions may be of particular relevance for the Russian Federation and other countries with a significant Slavic population.

Effect of Phlorotannins from Brown Algae Costaria costata on α-N-Acetylgalactosaminidase Produced by Duodenal Adenocarcinoma and Melanoma Cells.

The inhibitor of human α-N-acetylgalactosaminidase (α-NaGalase) was isolated from a water-ethanol extract of the brown algae Costaria costata. Currently, tumor α-NaGalase is considered to be a therapeutic target in the treatment of cancer. According to NMR spectroscopy and mass spectrometric analysis, it is a high-molecular-weight fraction of phlorethols with a degree of polymerization (DP) equaling 11-23 phloroglucinols (CcPh). It was shown that CcPh is a direct inhibitor of α-NaGalases isolated from HuTu 80 and SK-MEL-28 cells (IC50 0.14 ± 0.008 and 0.12 ± 0.004 mg/mL, respectively) and reduces the activity of this enzyme in HuTu 80 and SK-MEL-28 cells up to 50% at concentrations of 15.2 ± 9.5 and 5.7 ± 1.6 µg/mL, respectively. Molecular docking of the putative DP-15 oligophlorethol (P15OPh) and heptaphlorethol (PHPh) with human α-NaGalase (PDB ID 4DO4) showed that this compound forms a complex and interacts directly with the Asp 156 and Asp 217 catalytic residues of the enzyme in question. Thus, brown algae phlorethol CcPh is an effective marine-based natural inhibitor of the α-NaGalase of cancer cells and, therefore, has high therapeutic potential.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 α-N-acetylgalactosaminidase.

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α-N-acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O-glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N-acetylgalactosamine (GalNAc) from O-glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α-N-acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p-nitrophenyl α-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4C1 → 1S3 → 4C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

A missense variant in NDUFA6 confers schizophrenia risk by affecting YY1 binding and NAGA expression.

Genome-wide association studies (GWASs) have revealed that genetic variants at the 22q13.2 risk locus were robustly associated with schizophrenia. However, the causal variants at this risk locus and their roles in schizophrenia remain elusive. Here we identify the risk missense variant rs1801311 (located in the 1st exon of NDUFA6 gene) as likely causal for schizophrenia at 22q13.2 by disrupting binding of YY1, TAF1, and POLR2A. We systematically elucidated the regulatory mechanisms of rs1801311 and validated the regulatory effect of this missense variant. Intriguingly, rs1801311 physically interacted with NAGA (encodes the alpha-N-acetylgalactosaminidase, which is mainly involved in regulating metabolisms of glycoproteins and glycolipids in lysosome) and showed the most significant association with NAGA expression in the human brain, with the risk allele (G) associated with higher NAGA expression. Consistent with eQTL analysis, expression analysis showed that NAGA was significantly upregulated in brains of schizophrenia cases compared with controls, further supporting that rs1801311 may confer schizophrenia risk by regulating NAGA expression. Of note, we found that NAGA regulates important neurodevelopmental processes, including proliferation and differentiation of neural stem cells. Transcriptome analysis corroborated that NAGA regulates pathways associated with neuronal differentiation. Finally, we independently confirmed the association between rs1801311 and schizophrenia in a large Chinese cohort. Our study elucidates the regulatory mechanisms of the missense schizophrenia risk variant rs1801311 and provides mechanistic links between risk variant and schizophrenia etiology. In addition, this study also revealed the novel role of coding variants in gene regulation and schizophrenia risk, i.e., genetic variant in coding region of a specific gene may confer disease risk through regulating distal genes (act as regulatory variant for distal genes).

Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease.

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.

Biochemical characterization of Bombyx mori α-N-acetylgalactosaminidase belonging to the glycoside hydrolase family 31.

Horizontal gene transfer is an important evolutionary mechanism not only for bacteria but also for eukaryotes. In the domestic silkworm Bombyx mori, a model species of lepidopteran insects, some enzymes are known to have been acquired by horizontal transfer; however, the enzymatic features of protein BmNag31, belonging to glycoside hydrolase family 31 (GH31) and whose gene was predicted to be transferred from Enterococcus sp. are unknown. In this study, we reveal that the transcription of BmNag31 increases significantly during the prepupal to pupal stage, and decreases in the adult stage. The full-length BmNag31 and its truncated mutants were heterologously expressed in Escherichia coli and characterized. Its catalytic domain exhibits α-N-acetylgalactosaminidase activity and the carbohydrate-binding module family 32 domain shows binding activity towards N-acetylgalactosamine, similar to the Enterococcus faecalis homolog, EfNag31A. Gel filtration chromatography and blue native polyacrylamide gel electrophoresis analyses indicate that BmNag31 forms a hexamer whereas EfNag31A is monomeric. These results provide insights into the function of lepidopteran GH31 α-N-acetylgalactosaminidase.

Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening.

As part of a search for a substrate for droplet-based microfluidic screening assay of α-N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method for the challenging synthesis of coumarin glycosides of α-GalNAc was then developed and demonstrated with nine examples. The α-GalNAc glycoside of Jericho Blue prepared in this way was shown to function well under screening conditions.

Crystal structure of the Enterococcus faecalis α-N-acetylgalactosaminidase, a member of the glycoside hydrolase family 31.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.

Biochemical characterization of the endo-α-N-acetylgalactosaminidase pool of the human gut symbiont Tyzzerella nexilis.

Three large (2084-, 984-, and 2104-amino acids) endo-α-N-acetylgalactosaminidase candidate genes from the commensal human gut bacterium Tyzzerella nexilis were successfully cloned and subsequently expressed in Escherichia coli. Activity tests of the purified proteins revealed that two of the candidate genes (Tn0153 and Tn2105) were able to hydrolyze the disaccharide unit from Galß1-3GalNAc-α-pNP. The biochemical characterization revealed optimum pH conditions of 4.0 for both enzymes and temperature optima of 50 °C. The addition of 2-mercaptoethanol, Triton X-100 and urea had only minor effects on the activity of the enzymes, and the addition of imidazole and sodium dodecyl sulfate led to a significant reduction of the enzymes' activities. A mutational study identified and confirmed the role of the catalytically significant amino acids. The present study describes the first functional characterization of members of the GH101 family from this human gut symbiont.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

A Novel Homozygous Missense Variant in the NAGA Gene with Extreme Intrafamilial Phenotypic Heterogeneity.

Schindler disease is a rare autosomal recessive lysosomal storage disorder caused by a deficiency in alpha-N-acetylgalactosaminidase (α-NAGA) activity due to defects in the NAGA gene. Accumulation of the enzyme's substrates results in clinically heterogeneous symptoms ranging from asymptomatic individuals to individuals with severe neurological manifestations. Here, a 5-year-old Emirati male born to consanguineous parents presented with congenital microcephaly and severe neurological manifestations. Whole genome sequencing revealed a homozygous missense variant (c.838C>A; p.L280I) in the NAGA gene. The allele is a reported SNP in the ExAC database with a 0.0007497 allele frequency. The proband's asymptomatic sister and cousin carry the same genotype in a homozygous state as revealed from the family screening. Due to the extreme intrafamilial heterogeneity of the disease as seen in previously reported cases, we performed further analyses to establish the pathogenicity of this variant. Both the proband and his sister showed abnormal urine oligosaccharide patterns, which is consistent with the diagnosis of Schindler disease. The α-NAGA activity was significantly reduced in the proband and his sister with 5.9% and 12.1% of the mean normal activity, respectively. Despite the activity loss, p.L280I α-NAGA processing and trafficking were not affected. However, protein molecular dynamic simulation analysis revealed that this amino acid substitution is likely to affect the enzyme's natural dynamics and hinders its ability to bind to the active site. Functional analysis confirmed the pathogenicity of the identified missense variant and the diagnosis of Schindler disease. Extreme intrafamilial clinical heterogeneity of the disease necessitates further studies for proper genetic counseling and management.