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1.
J Diabetes Res ; 2024: 6942156, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38282657

RESUMO

Background: Better therapeutic drugs are required for treating hypertensive diabetic nephropathy. In our previous study, the Huaju Xiaoji (HJXJ) formula promoted the renal function of patients with diabetes and hypertensive nephropathy. In this study, we investigated the therapeutic effect and regulation mechanism of HJXJ in hypertensive diabetic mice with nephropathy. Methods: We constructed a mouse hypertensive diabetic nephropathy (HDN) model by treating mice with streptozotocin (STZ) and nomega-nitro-L-arginine methyl ester (LNAME). We also constructed a human glomerular mesangial cell (HGMC) model that was induced by high doses of sugar (30 mmol/mL) and TGFß1 (5 ng/mL). Pathological changes were evaluated by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and Masson staining. The fibrosis-related molecules (TGFß1, fibronectin, laminin, COL I, COL IV, α-SMA, and p-smad2/3) were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels and protein expression of endoplasmic reticulum stress, fibrosis molecules, and their downstream molecules were assessed using qPCR and Western blotting assays. Results: Administering HJXJ promoted the renal function of HDN mice. HJXJ reduced the expression of ER stress makers (CHOP and GRP78) and lncMGC, miR379, miR494, miR495, miR377, CUGBP2, CPEB4, EDEM3, and ATF3 in HDN mice and model HGMCs. The positive control drugs (dapagliflozin and valsartan) also showed similar effects after treatment with HJXJ. Additionally, in model HGMCs, the overexpression of CHOP or lncMGC decreased the effects of HJXJ-M on the level of fibrosis molecules and downstream target molecules. Conclusion: In this study, we showed that the HJXJ formula may regulate ERS-lncMGC/miRNA to enhance renal function in hypertensive diabetic mice with nephropathy. This study may act as a reference for further investigating whether combining HJXJ with other drugs can enhance its therapeutic effect. The findings of this study might provide new insights into the clinical treatment of hypertensive diabetic nephropathy with HJXJ.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Medicamentos de Ervas Chinesas , Hipertensão , MicroRNAs , Camundongos , Humanos , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , MicroRNAs/genética , MicroRNAs/uso terapêutico , Hipertensão/tratamento farmacológico , Modelos Animais de Doenças , Células Mesangiais/metabolismo , Fibrose , Proteínas de Ligação a RNA , Proteínas de Ligação ao Cálcio , alfa-Manosidase/metabolismo , alfa-Manosidase/uso terapêutico
2.
3.
Genes (Basel) ; 14(9)2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37761886

RESUMO

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.


Assuntos
Doenças por Armazenamento dos Lisossomos , alfa-Manosidose , Animais , Cães , alfa-Manosidase/genética , alfa-Manosidose/genética , alfa-Manosidose/veterinária , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/veterinária , Lisossomos , Mutação de Sentido Incorreto , Vacúolos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/veterinária
4.
ACS Chem Biol ; 18(8): 1730-1737, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37531094

RESUMO

Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor. Accordingly, SpGH92 is hypothesized to be a target for much-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→2)-ß-d-mannopyranoside (Manα1,2Manß-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manß-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.


Assuntos
Dissacarídeos , Streptococcus pneumoniae , Animais , Humanos , alfa-Manosidase/metabolismo , Fatores de Virulência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Polissacarídeos/metabolismo , Mamíferos/metabolismo
5.
Int J Biol Macromol ; 248: 126022, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37506790

RESUMO

Momordica charantia seeds contain a galactose specific lectin and mixture of glycosidases. These bind to lectin-affigel at pH 5.0 and are all eluted at pH 8.0. From the mixture, α-mannosidase was separated by gel filtration (purified enzyme Mr âˆ¼ 238 kDa). In native PAGE (silver staining) it showed three bands that stained with methylumbelliferyl substrate (possible isoforms). Ion exchange chromatography separated two isoforms in 0.5 M eluates and one isoform in 1.0 M eluate. In SDS-PAGE it dissociated to Mr ∼70 and 45 kDa subunits, showing antigenic similarity to jack bean enzyme. MALDI analysis confirmed the 70 kDa band to be α-mannosidase with sequence identity to the genomic sequence of Momordica charantia enzyme (score 83, 29 % sequence coverage). The pH, temperature optima were 5.0 and 60o C respectively. Kinetic parameters KM and Vmax estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC50 value was 50 nM). Secondary structural analysis at far UV (190-300 nm) showed 11.6 % α-helix and 36.5 % ß-sheets. 2.197 mg of the enzyme was found to interact with 3.75 mg of protein body membrane at pH 5.0 and not at pH 8.0 suggesting a pH dependent interaction.


Assuntos
Lectinas , Momordica charantia , alfa-Manosidase/química , Lectinas/metabolismo , Isoenzimas/metabolismo , Sementes/metabolismo
6.
Int J Surg ; 109(9): 2882-2885, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37352513

RESUMO

Alpha-mannosidosis (AM) is an autosomal recessive lysosomal storage disorder caused by reduced activity of the enzyme alpha-mannosidase. The disease is characterized by immunodeficiency, facial and skeletal abnormalities, impaired hearing, and intellectual disability. The clinical subtype of AM shows considerable variability in an individual, and at present, at least three clinical subtypes are suggested. Diagnosis is made by identification of deficiency of α-mannosidase activity in nucleated cells, like fibroblasts. The children are often born apparently normal as the disease is insidiously progressive, hence making early diagnosis essential. Along with supportive care, long-term therapeutic options include hematopoietic stem cell transplant, bone marrow transplantation, and enzyme replacement therapy. The possible benefits of these procedures must be weighed against the overall risk of procedure-related morbidity and mortality. Velmanase alfa is the first human recombinant form of alpha-mannosidase licensed and available for long-term enzyme replacement therapy. It is approved for treating non-neurologic manifestations of mild to moderate AM. The results obtained from different clinical trials provide evidence of the positive clinical effect of the recombinant enzyme on patients with AM. Different routes of diagnosis and unspecific initial symptoms of the disease lead to a delay in the initiation of treatment, resulting in accumulative morbidity. Thus, there is a dire necessity to create more awareness. Furthermore, additional multiple large-scale trials are needed to evaluate the long-term safety and efficacy of velmanase alfa.


Assuntos
alfa-Manosidose , Criança , Humanos , alfa-Manosidose/terapia , alfa-Manosidose/tratamento farmacológico , alfa-Manosidase/uso terapêutico , Transplante de Medula Óssea , Cognição , Terapia de Reposição de Enzimas
7.
Microbiol Spectr ; 11(3): e0482422, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37154721

RESUMO

Endophytes play an important role in shaping plant growth and immunity. However, the mechanisms for endophyte-induced disease resistance in host plants remain unclear. Here, we screened and isolated the immunity inducer ShAM1 from the endophyte Streptomyces hygroscopicus OsiSh-2, which strongly antagonizes the pathogen Magnaporthe oryzae. Recombinant ShAM1 can trigger rice immune responses and induce hypersensitive responses in various plant species. After infection with M. oryzae, blast resistance was dramatically improved in ShAM1-inoculated rice. In addition, the enhanced disease resistance by ShAM1 was found to occur through a priming strategy and was mainly regulated through the jasmonic acid-ethylene (JA/ET)-dependent signaling pathway. ShAM1 was identified as a novel α-mannosidase, and its induction of immunity is dependent on its enzyme activity. When we incubated ShAM1 with isolated rice cell walls, the release of oligosaccharides was observed. Notably, extracts from the ShAM1-digested cell wall can enhance the disease resistance of the host rice. These results indicated that ShAM1 triggered immune defense against pathogens by damage-associated molecular pattern (DAMP)-related mechanisms. Our work provides a representative example of endophyte-mediated modulation of disease resistance in host plants. The effects of ShAM1 indicate the promise of using active components from endophytes as plant defense elicitors for the management of plant disease. IMPORTANCE The specific biological niche inside host plants allows endophytes to regulate plant disease resistance effectively. However, there have been few reports on the role of active metabolites from endophytes in inducing host disease resistance. In this study, we demonstrated that an identified α-mannosidase protein, ShAM1, secreted by the endophyte S. hygroscopicus OsiSh-2 could activate typical plant immunity responses and induce a timely and cost-efficient priming defense against the pathogen M. oryzae in rice. Importantly, we revealed that ShAM1 enhanced plant disease resistance through its hydrolytic enzyme (HE) activity to digest the rice cell wall and release damage-associated molecular patterns. Taken together, these findings provide an example of the interaction mode of endophyte-plant symbionts and suggest that HEs derived from endophytes can be used as environmentally friendly and safe prevention agent for plant disease control.


Assuntos
Magnaporthe , Oryza , Resistência à Doença , Endófitos/fisiologia , alfa-Manosidase/metabolismo , alfa-Manosidase/farmacologia , Magnaporthe/metabolismo , Doenças das Plantas , Parede Celular
8.
Plant Mol Biol ; 112(3): 107-117, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37178231

RESUMO

Cell wall is a strong and complex net whose function is to provide turgor, pathogens attack protection and to give structural support to the cell. In growing and expanding cells, the cell wall of fruits is changing in space and time, because they are changing according to stage of ripening. Understand which mechanisms to produce significant could help to develop tools to prolong the fruit shelf life. Cell wall proteins (CWPs) with enzymatic activity on cell wall polysaccharides, have been studied widely. Another investigations take place in the study of N-glycosylations of CWPs and enzymes with activity on glycosidic linkages. α-mannosidase (α-Man; EC 3.2.1.24) and ß-D-N-acetylhexosaminidase (ß-Hex; EC 3.2.1.52), are enzymes with activity on mannose and N-acetylglucosamine sugar presents in proteins as part of N-glycosylations. Experimental evidence indicate that both are closely related to loss of fruit firmness, but in the literature, there is still no review of both enzymes involved fruit ripening. This review provides a complete state-of-the-art of α-Man and ß-Hex enzymes related in fruit ripening. Also, we propose a vesicular α-Man (EC 3.2.1.24) name to α-Man involved in N-deglycosylations of CWPs of plants.


Assuntos
Frutas , Glicosídeo Hidrolases , alfa-Manosidase/metabolismo , Glicosídeo Hidrolases/metabolismo , Frutas/metabolismo , Polissacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Parede Celular/metabolismo
9.
Glycobiology ; 33(9): 687-699, 2023 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-37202179

RESUMO

While glycans underlie many biological processes, such as protein folding, cell adhesion, and cell-cell recognition, deep evolution of glycosylation machinery remains an understudied topic. N-linked glycosylation is a conserved process in which mannosidases are key trimming enzymes. One of them is the glycoprotein endo-α-1,2-mannosidase which participates in the initial trimming of mannose moieties from an N-linked glycan inside the cis-Golgi. It is unique as the only endo-acting mannosidase found in this organelle. Relatively little is known about its origins and evolutionary history; so far it was reported to occur only in vertebrates. In this work, a taxon-rich bioinformatic survey to unravel the evolutionary history of this enzyme, including all major eukaryotic clades and a wide representation of animals, is presented. The endomannosidase was found to be more widely distributed in animals and other eukaryotes. The protein motif changes in context of the canonical animal enzyme were tracked. Additionally, the data show the two canonical vertebrate endomannosidase genes, MANEA and MANEAL, arose at the second round of the two vertebrate genome duplications and one more vertebrate paralog, CMANEAL, is uncovered. Finally, a framework where N-glycosylation co-evolved with complex multicellularity is described. A better understanding of the evolution of core glycosylation pathways is pivotal to understanding biology of eukaryotes in general, and the Golgi apparatus in particular. This systematic analysis of the endomannosidase evolution is one step toward this goal.


Assuntos
Manosidases , Polissacarídeos , Animais , alfa-Manosidase/genética , alfa-Manosidase/metabolismo , Filogenia , Manosidases/genética , Manosidases/metabolismo , Polissacarídeos/metabolismo , Glicosilação , Vertebrados/metabolismo , Eucariotos/metabolismo , Complexo de Golgi/metabolismo
10.
Int J Pediatr Otorhinolaryngol ; 169: 111556, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37099947

RESUMO

Alpha-mannosidase catalyze lysosomal cleaving of mannose residues from glycoproteins. The enzyme is encoded by the MAN2B1 gene. Biallelic pathogenic variants cause enzymatic deficiency, which clinically results in alpha-mannosidosis (AM), an autosomal recessively inherited condition. Typical features observed in AM patients include intellectual disability, loss of speech, dysmorphic features, progressive motor problems, ataxia, hearing impairment and recurrent otitis. The cause of the latter is mainly attributed to immunodeficiency. The aim of our study was to demonstrate the otolaryngologic and hearing outcomes in patients with AM. The study group consisted of 8 AM patients: 6 males and 2 females, aged 2.5-37 yrs. The clinical course, dysmorphic ENT features, hearing status and the HRCT scans of the temporal bones were analyzed. MS Excel for Windows and Statistica software package were used for the comparison of interaural audiometric loss, mean hearing loss and mean hearing threshold for each patient's audiometric frequency tested. We identified ENT dysmorphic features in all of our AM patients, while the hearing loss was detected in 6 out of our 8 patients. For those cases, the onset of deafness was noted in the first decade of life, this impairment was sensorineural, of cochlear origin, bilateral, of a moderate degree (mean loss 62.76 dB; median 60 dB, standard deviation 12.5 dB), symmetrical and stable. The shape of the audiometric curves of our patients can be described as slightly sloping towards the higher tested frequencies, with a marked improvement at 4 kHz. The radiological examination revealed normal structures of the ears, with the exception of one case where a persistent otitis generated a cochlear gap. We therefore concluded that the hearing loss in our AM patients derived from cochlear impairment unrelated with recurrent otitis.


Assuntos
Perda Auditiva Neurossensorial , Perda Auditiva , alfa-Manosidose , Masculino , Feminino , Humanos , alfa-Manosidose/diagnóstico por imagem , alfa-Manosidose/genética , alfa-Manosidose/patologia , Polônia , Perda Auditiva/diagnóstico por imagem , Perda Auditiva/genética , alfa-Manosidase/química , alfa-Manosidase/genética , Audiometria
11.
Acta Crystallogr D Struct Biol ; 79(Pt 5): 387-400, 2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-37071393

RESUMO

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.


Assuntos
Mananas , Manosidases , Manosidases/química , Manosidases/metabolismo , alfa-Manosidase/metabolismo , Mananas/química , Mananas/metabolismo , Manose/química , Manose/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Polissacarídeos/química , Especificidade por Substrato
12.
J Inherit Metab Dis ; 46(4): 705-719, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36849760

RESUMO

Alpha-mannosidosis (AM) is a rare, autosomal recessive, lysosomal storage disorder caused by alpha-mannosidase deficiency that leads to the accumulation of mannose-rich oligosaccharides. AM symptoms and severity vary among individuals; consequently, AM is often not diagnosed until late childhood. Velmanase alfa (VA), a recombinant human lysosomal alpha-mannosidase product, is the first enzyme replacement therapy indicated to treat non-neurological symptoms of AM in Europe. Previous studies suggested that early VA treatment in children may produce greater clinical benefit over the disease course than starting treatment in adolescents or adults; however, long-term studies in children are limited, and very few studies include children under 6 years of age. The present phase 2, multicenter, open-label study evaluated the safety and efficacy of long-term VA treatment in children under 6 years of age with AM. Five children (three males) received VA weekly for ≥24 months, and all children completed the study. Four children experienced adverse drug reactions (16 events) and two experienced infusion-related reactions (12 events). Most (99.5%) adverse events were mild or moderate, and none caused study discontinuation. Four children developed antidrug antibodies (three were neutralizing). After VA treatment, all children improved in one or more efficacy assessments of serum oligosaccharide concentrations (decreases), hearing, immunological profile, and quality of life, suggesting a beneficial effect of early treatment. Although the small study size limits conclusions, these results suggest that long-term VA treatment has an acceptable safety profile, is well tolerated, and may provide potential benefits to patients with AM under 6 years of age.


Assuntos
alfa-Manosidose , Masculino , Adulto , Adolescente , Humanos , Criança , Pré-Escolar , Qualidade de Vida , alfa-Manosidase/efeitos adversos , Lisossomos , Anticorpos
13.
Bioorg Chem ; 132: 106373, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36681043

RESUMO

Synthetic glycoconjugates as chemical probes have been widely developed for the detection of glycosidase enzymes. However, the binding interactions between iminosugar derivatives and glycosidases were limited, especially for the binding interactions between multivalent glycosidase inhibitors and α-glycosidases. In this paper, three naphthalimide-DNJ conjugates were synthesized. Furthermore, the binding interactions and glycosidase inhibition effects of them were investigated. It was found that the strong binding interactions of multivalent glycosidase inhibitors with enzymes were related to the efficient inhibitory activity against glycosidase. Moreover, the lengths of the chain between DNJ moieties and the triazole ring for the naphthalimide-DNJ conjugates influenced the self-assembly properties, binding interactions and glycosidase inhibition activities with multisource glycosidases. Compound 13 with six carbons between the DNJ moiety and triazole ring showed the stronger binding interactions and better glycosidase inhibition activities against α-mannosidase (jack bean) and α-glucosidase (aspergillus niger). In addition, compound 13 showed an effective PBG inhibition effect in mice with 51.18 % decrease in blood glucose at 30 min. This result opens a way for detection of multivalent glycosidase inhibition effect by a fluorescent sensing method.


Assuntos
Inibidores Enzimáticos , Glicosídeo Hidrolases , Camundongos , Animais , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Naftalimidas/farmacologia , Fluorescência , alfa-Manosidase
14.
J Vet Med Sci ; 85(2): 244-251, 2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36596563

RESUMO

Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAMM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.


Assuntos
Lisossomos , Swainsonina , Animais , alfa-Manosidase/genética , Simulação de Acoplamento Molecular , Lisossomos/metabolismo , Cabras/metabolismo , Manosidases/metabolismo
15.
Appl Biochem Biotechnol ; 195(3): 1823-1836, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36399304

RESUMO

This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0-6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co2+ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu2+, Zn2+, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins.


Assuntos
Bactérias , Manosidases , alfa-Manosidase/genética , alfa-Manosidase/química , Bactérias/metabolismo , Cinética , Manosidases/metabolismo
16.
Org Biomol Chem ; 20(45): 8932-8943, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36322142

RESUMO

The development of effective inhibitors of Golgi α-mannosidase II (GMII, E.C.3.2.1.114) with minimal off-target effects on phylogenetically-related lysosomal α-mannosidase (LMan, E.C.3.2.1.24) is a complex task due to the complicated structural and chemical properties of their active sites. The pKa values (and also protonation forms in some cases) of several ionizable amino acids, such as Asp, Glu, His or Arg of enzymes, can be changed upon the binding of the inhibitor. Moreover, GMII and LMan work under different pH conditions. The pKa calculations on large enzyme-inhibitor complexes and FMO-PIEDA energy decomposition analysis were performed on the structures of selected inhibitors obtained from docking and hybrid QM/MM calculations. Based on the calculations, the roles of the amino group incorporated in the ring of the imino-D-lyxitol inhibitors and some ionizable amino acids of Golgi-type (Asp270-Asp340-Asp341 of Drosophila melanogaster α-mannosidase dGMII) and lysosomal-type enzymes (His209-Asp267-Asp268 of Canavalia ensiformis α-mannosidase, JBMan) were explained in connection with the observed inhibitory properties. The pyrrolidine ring of the imino-D-lyxitols prefers at the active site of dGMII the neutral form while in JBMan the protonated form, whereas that of imino-L-lyxitols prefers the protonation form in both enzymes. The calculations indicate that the binding mechanism of inhibitors to the active-site of α-mannosidases is dependent on the inhibitor structure and could be used to design new selective inhibitors of GMII. A series of novel synthetic N-substituted imino-D-lyxitols were evaluated with four enzymes from the glycoside hydrolase GH38 family (two of Golgi-type, Drosophila melanogaster GMIIb and Caenorhabditis elegans AMAN-2, and two of lysosomal-type, Drosophila melanogaster LManII and Canavalia ensiformis JBMan, enzymes). The most potent structures [N-9-amidinononyl and N-2-(1-naphthyl)ethyl derivatives] inhibited GMIIb (Ki = 40 nM) and AMAN-2 (Ki = 150 nM) with a weak selectivity index (SI) toward Golgi-type enzymes of IC50(LManII)/IC50(GMIIb) = 35 or IC50(JBMan)/IC50(AMAN-2) = 86. On the other hand, weaker micromolar inhibitors, such as N-2-naphthylmethyl or 4-iodobenzyl derivatives [IC50(GMIIb) = 2.4 µM and IC50 (AMAN-2) = 7.6 µM], showed a significant SI in the range from 111 to 812.


Assuntos
Drosophila melanogaster , Manosidases , Animais , alfa-Manosidase/química , Drosophila melanogaster/metabolismo , Manosidases/química , Manosidases/metabolismo , Inibidores Enzimáticos/química , Aminoácidos , Amantadina
17.
Proc Natl Acad Sci U S A ; 119(39): e2208168119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122227

RESUMO

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.


Assuntos
Bacteroides , Glicosídeo Hidrolases , Glicosídeo Hidrolases/química , Humanos , Plantas/metabolismo , Polissacarídeos/metabolismo , Açúcares/metabolismo , alfa-Manosidase/metabolismo
18.
J Biol Chem ; 298(9): 102313, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35921895

RESUMO

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell-pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected ß1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel ß1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal ß1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.


Assuntos
Polissacarídeos , Xanthomonas , alfa-Manosidase , beta-Galactosidase , Galactose/metabolismo , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Manose , Manosídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Xanthomonas/enzimologia , alfa-Manosidase/metabolismo , beta-Galactosidase/metabolismo
19.
Int J Mol Sci ; 23(15)2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35897761

RESUMO

Prostate cancer is the most common cancer in men, and it is primarily driven by androgen steroid hormones. The glycosylation enzyme EDEM3 is controlled by androgen signalling and is important for prostate cancer viability. EDEM3 is a mannosidase that trims mannose from mis-folded glycoproteins, tagging them for degradation through endoplasmic reticulum-associated degradation. Here, we find that EDEM3 is upregulated in prostate cancer, and this is linked to poorer disease-free survival. Depletion of EDEM3 from prostate cancer cells induces an ER stress transcriptomic signature, and EDEM3 overexpression is cyto-protective against ER stressors. EDEM3 expression also positively correlates with genes involved in the unfolded protein response in prostate cancer patients, and its expression can be induced through exposure to radiation. Importantly, the overexpression of EDEM3 promotes radio-resistance in prostate cancer cells and radio-resistance can be reduced through depletion of EDEM3. Our data thus implicate increased levels of EDEM3 with a role in prostate cancer pathology and reveal a new therapeutic opportunity to sensitise prostate tumours to radiotherapy.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Neoplasias da Próstata , Androgênios/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Masculino , Manosidases/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , alfa-Manosidase/metabolismo
20.
Orphanet J Rare Dis ; 17(1): 287, 2022 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-35871018

RESUMO

BACKGROUND: Alpha-mannosidosis is a rare autosomal recessive lysosomal storage disorder (LSD) caused by reduced activity of alpha-mannosidase. Clinical manifestations include skeletal dysmorphism, mental impairment, hearing loss and recurrent infections. The severe type of the disease leads to early childhood death, while patients with milder forms can live into adulthood. There are no mortality studies to date. This study aimed to investigate the age at death and the causes of death of patients with alpha-mannosidosis who had not received disease-modifying treatment. METHODS: Clinicians and LSD patient organisations (POs) from 33 countries were invited to complete a questionnaire between April-May 2021. Cause of death and age at death was available for 15 patients. A literature review identified seven deceased patients that met the inclusion criteria. RESULTS: Median age at death for patients reported by clinicians/POs was 45 years (mean 40.3 ± 13.2, range 18-56, n = 15); 53% were female. One death occurred during the patient's second decade of life, and 14 out of 15 deaths (93.3%) during or after the patients' third decade, including four (26.7%) during their sixth decade. Median age at death for patients identified from the literature was 4.3 years (mean 15.7 ± 17.0, range 2.2-41, n = 7); two were female. Four of the seven patients (57.1%) died within the first decade of life. Seven of 15 deaths (46.7%) reported by clinicians/POs were recorded as pneumonia and three (20.0%) as cancer. Other causes of death included acute renal failure due to sepsis after intestinal perforation, decrease of red blood cells of unknown origin, kidney failure with systemic lupus erythematosus, aortic valve insufficiency leading to heart failure, and dehydration due to catatonia. Three out of seven causes of death (42.9%) reported in the literature were associated with septicaemia, two (28.6%) with respiratory failure and one to pneumonia following aspiration. CONCLUSIONS: This study suggests that pneumonia has been the primary cause of death during recent decades in untreated patients with alpha-mannosidosis, followed by cancer. Determining the causes of mortality and life expectancy in these patients is crucial to further improve our understanding of the natural history of alpha-mannosidosis.


Assuntos
Perda Auditiva , Deficiência Intelectual , alfa-Manosidose , Adulto , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , alfa-Manosidase
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