Genetic features of patients with MPS type IIIB: Description of five pathogenic gene variations.

BACKGROUND: There are four distinct forms of Sanfilippo syndrome (MPS type III), each of which is an autosomal lysosomal storage disorder. These forms are caused by abnormalities in one of four lysosomal enzymes. This study aimed to identify possible genetic variants that contribute to Sanfilippo IIIB in 14 independent families in Southwest Iran. METHODS: Patients were included if their clinical features and enzyme assay results were suggestive. The patients were subsequently subjected to Sanger Sequencing to screen for Sanfilippo-related genes. Additional investigations have been conducted using various computational analyses to determine the probable functional effects of diagnosed variants. RESULTS: Five distinct variations were identified in the NAGLU gene. This included two novel variants in two distinct families and three previously reported variants in 12 distinct families. All of these variations were recognized as pathogenic using the MutationTaster web server. In silico analysis showed that all detected variants affected protein structural stability; four destabilized protein structures, and the fifth variation had the opposite effect. CONCLUSION: In this study, two novel variations in the NAGLU gene were identified. The results of this study positively contribute to the mutation diversity of the NAGLU gene. To identify new disease biomarkers and therapeutic targets, precision medicine must precisely characterize and account for genetic variations. New harmful gene variants are valuable for updating gene databases concerning Sanfilippo disease variations and NGS gene panels. This may also improve genetic counselling for rapid risk examinations and disease surveillance.

Disease pathology signatures in a mouse model of Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare and devastating childhood-onset lysosomal storage disease caused by complete loss of function of the lysosomal hydrolase α-N-acetylglucosaminidase. The lack of functional enzyme in MPS IIIB patients leads to the progressive accumulation of heparan sulfate throughout the body and triggers a cascade of neuroinflammatory and other biochemical processes ultimately resulting in severe mental impairment and early death in adolescence or young adulthood. The low prevalence and severity of the disease has necessitated the use of animal models to improve our knowledge of the pathophysiology and for the development of therapeutic treatments. In this study, we took a systematic approach to characterizing a classical mouse model of MPS IIIB. Using a series of histological, biochemical, proteomic and behavioral assays, we tested MPS IIIB mice at two stages: during the pre-symptomatic and early symptomatic phases of disease development, in order to validate previously described phenotypes, explore new mechanisms of disease pathology and uncover biomarkers for MPS IIIB. Along with previous findings, this study helps provide a deeper understanding of the pathology landscape of this rare disease with high unmet medical need and serves as an important resource to the scientific community.

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

Construction of tomato plants with suppressed endo-ß-N-acetylglucosaminidase activity using CRISPR-Cas9 mediated genome editing.

High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-ß-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Δeng mutants (T0 and T1 generations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Δeng1-2, -22, and -26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Δeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Δeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Δeng mutants (T2 generation) also showed loss of ENGase activity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Δeng mutants and the wild type. The Δeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.

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.

Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Schizophreniform presentation and abrupt neurologic decline in a patient with late-onset mucopolysaccharidosis type IIIB.

Due to their low frequency and some atypical presentations, inborn errors of metabolism are frequently misdiagnosed or underdiagnosed, which hinders the correct management of these patients. To illustrate that, here we present a patient that, at early school age, had learning disabilities compared to her classmates, especially for writing. She completed basic education in a regular school and was transferred to a secondary school for students with special needs. At 18 years of age, she presented a first psychiatric abrupt outbreak: she spent a month screaming and without sleeping. Behavioral problems then became apparent, especially hyperactivity, destructive and chaotic behavior, anxiety, and auto-aggressivity and hetero-aggressivity. A diagnosis of schizophreniform disorder was established. Clinical genetic evaluation revealed coarse face, macroglossia, coarse thick hair, and mild hepatomegaly, and the hypothesis of mucopolysaccharidosis-III was raised. Laboratory tests indicated high levels of urinary glycosaminoglycans and almost undetectable NAGLU activity, confirming the diagnosis. Sequencing of the NAGLU gene revealed the c.1318G>C (p.Gly440Arg) and c.1834A>G (p.Ser612Gly) mutations.

Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.

Significant neuropsychiatric symptoms: three mucopolysaccharidosis type IIIB cases, two of whom were siblings with a novel NAGLU gene mutation.

Mucopolysaccharidosis (MPS) type IIIB patients present with marked neurodevelopmental and neuropsychiatric problems and not with typical MPS symptoms such as coarse facial features, organomegaly, or short body height, especially at the first presentation. We present three pediatric cases, two of which are sisters with novel NAGLU gene mutations, to emphasize that diagnosis of MPS type IIIB should be remembered in patients presenting with neurodevelopmental and neuropsychiatric problems such as delayed speech, autistic-like symptoms, severe behavioral and sleep problems, motor deterioration or idiopathic intellectual disability with or without refractory epilepsy, especially if there is aconsanguineous marriage.

Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome.

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients' CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells.

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese hamster ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with nine different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

Biochemical evaluation of intracerebroventricular rhNAGLU-IGF2 enzyme replacement therapy in neonatal mice with Sanfilippo B syndrome.

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu-/- neonatal mice. Twelve weeks after treatment, NAGLU activity levels in brain were 0.75-fold those of controls. HS and ß-hexosaminidase activity, which are elevated in MPS IIIB, decreased to normal levels. This effect persisted for at least 4 weeks after treatment. Elevated NAGLU and reduced ß-hexosaminidase activity levels were detected in liver; these effects persisted for up to 4 weeks after treatment. The overall therapeutic effects of single dose ICV-ERT with rhNAGLU-IGF2 in Naglu-/- neonatal mice were long-lasting. These results suggest a potential benefit of early treatment, followed by less-frequent ICV-ERT dosing, in patients diagnosed with MPS IIIB.

Induced pluripotent stem cell line (SDQLCHi041-A) from a male patient with mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive metabolic disorder caused by mutations in NAGLU gene, and characterized by progressive cognitive decline and behavioral difficulties and motor function retardation. A human induced pluripotent stem cell line, SDQLCHi041-A was generated from peripheral blood mononuclear cells of a 4 years and 9 months old patient with MPS IIIB, who carried compound heterozygous mutation of c.1336G > A and c.608G > A in NAGLU gene. SDQLCHi041-A offers a useful cell model to investigate pathogenic mechanisms in MPS IIIB.

Characterization and functional analysis of chitinase family genes involved in nymph-adult transition of Sogatella furcifera.

Chitinase degrades chitin in the old epidermis or peritrophic matrix of insects, which ensures normal development and metamorphosis. In our previous work, we comprehensively studied the function of SfCht7 in Sogatella furcifera. However, the number and function of chitinase genes in S. furcifera remain unknown. Here, we identified 12 full-length chitinase transcripts from S. furcifera, which included nine chitinase (Cht), two imaginal disc growth factor (IDGF), and one endo-ß-N-acetylglucosaminidase (ENGase) genes. Expression analysis results revealed that the expression levels of eight genes (SfCht3, SfCht5, SfCht6-1, SfCht6-2, SfCht7, SfCht8, SfCht10, and SfIDGF2) with similar transcript levels peaked prior to molting of each nymph and were highly expressed in the integument. Based on RNA interference (RNAi), description of the functions of each chitinase gene indicated that the silencing of SfCht5, SfCht10, and SfIDGF2 led to molting defects and lethality. RNAi inhibited the expressions of SfCht5, SfCht7, SfCht10, and SfIDGF2, which led to downregulated expressions of chitin synthase 1 (SfCHS1, SfCHS1a, and SfCHS1b) and four chitin deacetylase genes (SfCDA1, SfCDA2, SfCDA3, and SfCDA4), and caused a change in the expression level of two trehalase genes (TRE1 and TRE2). Furthermore, silencing of SfCht7 induced a significant decrease in the expression levels of three wing development-related genes (SfWG, SfDpp, and SfHh). In conclusion, SfCht5, SfCht7, SfCht10, and SfIDGF2 play vital roles in nymph-adult transition and are involved in the regulation of chitin metabolism, and SfCht7 is also involved in wing development; therefore, these genes are potential targets for control of S. furcifera.

Coinheritance of novel mutations in NAGLU causing mucopolysaccharidosis type IIIB and in DDHD2 causing spastic paraplegia54 in a Turkish family.

Mucopolysaccharidosis type IIIB (MPSIIIB) is one of the lysosomal storage diseases, clinically related to developmental delay in the early phase and loss of skills in the late phases of the disease. The disease is caused by homozygous mutations in the NAGLU gene. Spastic paraplegia54 (SPG54) is a neurodegenerative disorder caused by homozygous mutations in the DDHD2 gene. Clinical features are progressive spasticity and weakness in the lower limbs and corpus callosum agenesis. We report on two siblings in a consanguineous family, presenting both the clinical and molecular diagnoses of MPSIIIB and SPG54 with novel mutations by using whole exome sequencing (WES). This interesting finding shows that we should be aware of the importance of using WES for diagnosing rare diseases in consanguineous families.

Structural insights of the enzymes from the chitin utilization locus of Flavobacterium johnsoniae.

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (ß-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase.

Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-ß-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs.

The beta-1, 4-N-acetylglucosaminidase 1 gene, selected by domestication and breeding, is involved in cocoon construction of Bombyx mori.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that ß-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.