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1.
Cell Mol Life Sci ; 79(7): 352, 2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35676564

RESUMO

Immune checkpoint blockade therapy has drastically improved the prognosis of certain advanced-stage cancers. However, low response rates and immune-related adverse events remain important limitations. Here, we report that inhibiting ALG3, an a-1,3-mannosyltransferase involved in protein glycosylation in the endoplasmic reticulum (ER), can boost the response of tumors to immune checkpoint blockade therapy. Deleting N-linked glycosylation gene ALG3 in mouse cancer cells substantially attenuates their growth in mice in a manner depending on cytotoxic T cells. Furthermore, ALG3 inhibition or N-linked glycosylation inhibitor tunicamycin treatment synergizes with anti-PD1 therapy in suppressing tumor growth in mouse models of cancer. Mechanistically, we found that inhibiting ALG3 induced deficiencies of post-translational N-linked glycosylation modification and led to excessive lipid accumulation through sterol-regulated element-binding protein (SREBP1)-dependent lipogenesis in cancer cells. N-linked glycosylation deficiency-mediated lipid hyperperoxidation induced immunogenic ferroptosis of cancer cells and promoted a pro-inflammatory microenvironment, which boosted anti-tumor immune responses. In human subjects with cancer, elevated levels of ALG3 expression in tumor tissues are associated with poor patient survival. Taken together, we reveal an unappreciated role of ALG3 in regulating tumor immunogenicity and propose a potential therapeutic strategy for enhancing cancer immunotherapy.


Assuntos
Ferroptose , Manosiltransferases , Neoplasias , Animais , Humanos , Inibidores de Checkpoint Imunológico , Imunoterapia , Lipídeos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Camundongos , Neoplasias/terapia
2.
ACS Infect Dis ; 8(6): 1116-1123, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-35594144

RESUMO

C-Mannosylation of the thrombospondin type I repeat (TSR) domains is one of the most important factors involved in their function. It occurs on the first tryptophan of the WXXWXXC conserved motif where the tryptophan is usually surrounded by arginine or lysine forming the ligand-binding stretch of this sticky domain. It is found in its canonical or modified forms in many Plasmodium proteins. TSR containing proteins such as thrombospondin-like anonymous protein (TRAP), circumsporozoite protein (CSP), CSP and TRAP related protein (CTRP), and secreted protein with altered thrombospondin repeat (SPATR) have all been shown to be important for various parasite processes and life cycle stages. Here, we show that C-mannosylation catalyzing enzyme C-mannosyltransferase (CmanT) plays an essential role in malaria transmission in Plasmodium berghei. Disruption of the CmanT does not affect asexual blood stage propagation or gametocyte development but abolishes the formation of oocysts in mosquitoes. CmanT knockout (CmanT-) parasites showed normal ookinete formation; however, these ookinetes failed in their ability to glide. CmanT- was complemented by reintroducing the gene, restoring mosquito transmission to wild-type level. We also investigated the effect of C-mannosylation on the folding and heparin-binding capacity of the Plasmodium falciparum TRAP TSR domain in silico, which suggested that this phenotype should be due to its involvement in the global stabilization of TSR residue side chain interactions.


Assuntos
Culicidae , Malária , Animais , Malária/parasitologia , Manosiltransferases/genética , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Triptofano
3.
Dev Biol ; 486: 96-108, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35367457

RESUMO

Skin epidermis secretes apical extracellular matrix (aECM) as a protective barrier from the external environment. The aECM is highly dynamic and constantly undergoes remodeling during animal development. How aECM dynamics is temporally regulated during development, and whether and how its mis-regulation may impact epidermal cell morphology or function remains to be fully elucidated. Here, we report that the conserved Zn-finger transcription factor BLMP-1/Blimp1, which regulates epidermal development in C. elegans, controls apical cell shape of the epidermis by downregulation of aECM remodeling. Loss of blmp-1 causes upregulation of genes essential for molting, including bus-8 and mlt-8, in adult, leading to an abnormal shape in the apical region of adult epidermal cells. The apical epidermal morphological defect is suppressed by reduction of bus-8 or mlt-8. BUS-8 is a key mannosyltransferase, which functions in glycosylation of N-linked glycoproteins; MLT-8 has a ganglioside GM2 lipid-binding domain and is implicated in signaling during molting, a process where the old cuticle is shed and synthesized anew. Overexpression of bus-8 or mlt-8 induces an apical epidermal cell defect as observed in blmp-1 mutants. MLT-8::GFP fusion protein is localized to lysosomes and secreted to aECM. BUS-8 is important for MLT-8 stability and lysosomal targeting, which may be regulated by BUS-8-mediated glycosylation of MLT-8 and function as a molting signaling cue in aECM remodeling. We propose that BLMP-1 represses MLT-8 expression and glycosylation in the epidermis to prevent inappropriate aECM remodeling, which is essential for maintenance of apical epidermal cell morphology during larva-to-adult transition.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Manosiltransferases/metabolismo , Muda/genética
4.
J Inherit Metab Dis ; 45(4): 769-781, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35279850

RESUMO

Congenital disorders of glycosylation type 1 (CDG-I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG-I is a partial absence of complete N-glycans on transferrin. However, recent findings of a diagnostic N-tetrasaccharide for ALG1-CDG and increased high-mannose N-glycans for a few other CDG suggested the potential of glycan structural analysis for CDG-I gene discovery. We analyzed the relative abundance of total plasma N-glycans by high resolution quadrupole time-of-flight mass spectrometry in a large cohort of 111 CDG-I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single-molecule molecular inversion probes (smMIPs) for sequencing of CDG-I candidate genes on the basis of specific N-glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N-tetrasaccharide in ALG2-CDG patients and a novel fucosylated N-pentasaccharide as specific glycomarker for ALG1-CDG. Moreover, group-specific high-mannose N-glycan signatures were found in ALG3-, ALG9-, ALG11-, ALG12-, RFT1-, SRD5A3-, DOLK-, DPM1-, DPM3-, MPDU1-, ALG13-CDG, and hereditary fructose intolerance. Further differential analysis revealed high-mannose profiles, characteristic for ALG12- and ALG9-CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG-I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG-I patient cohorts.


Assuntos
Defeitos Congênitos da Glicosilação , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Glicômica , Glicosilação , Humanos , Manose , Manosiltransferases/genética , N-Acetilglucosaminiltransferases , Oligossacarídeos , Polissacarídeos/genética
5.
Genetics ; 221(1)2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35333306

RESUMO

Candida albicans cell wall glycoproteins, and in particular their mannose-rich glycans, are important for maintaining cellular integrity as well as host recognition, adhesion, and immunomodulation. The asparagine (N)-linked mannose outer chain of these glycoproteins is produced by Golgi mannosyltransferases (MTases). The outer chain is composed of a linear backbone of ∼50 α1,6-linked mannoses, which acts as a scaffold for addition of ∼150 or more mannoses in other linkages. Here, we describe the characterization of C. albicans OCH1, MNN9, VAN1, ANP1, MNN10, and MNN11, which encode the conserved Golgi MTases that sequentially catalyze the α1,6 mannose outer chain backbone. Candida albicans och1Δ/Δ, mnn9Δ/Δ, and van1Δ/Δ mutants block the earliest steps of backbone synthesis and like their Saccharomyces cerevisiae counterparts, have severe cell wall and growth phenotypes. Unexpectedly, and in stark contrast to S. cerevisiae, loss of Anp1, Mnn10, or Mnn11, which together synthesize most of the backbone, have no obvious deleterious phenotypes. These mutants were unaffected in cell morphology, growth, drug sensitivities, hyphal formation, and macrophage recognition. Analyses of secreted glycosylation reporters demonstrated that anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ strains accumulate glycoproteins with severely truncated N-glycan chains. This hypo-mannosylation did not elicit increased chitin deposition in the cell wall, which in other yeast and fungi is a key compensatory response to cell wall integrity breaches. Thus, C. albicans has evolved an alternate mechanism to adapt to cell wall weakness when N-linked mannan levels are reduced.


Assuntos
Candida albicans , Parede Celular , Manosiltransferases , Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Candida albicans/citologia , Candida albicans/genética , Candida albicans/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Manose/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Commun Biol ; 5(1): 117, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136180

RESUMO

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.


Assuntos
Manosiltransferases , Proteínas de Saccharomyces cerevisiae , Vias Biossintéticas , Dolicóis/metabolismo , Humanos , Lipopolissacarídeos , Manosiltransferases/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Mol Cell ; 82(3): 585-597.e11, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35120648

RESUMO

Cullin-RING E3 ligases (CRLs) are essential ubiquitylation enzymes that combine a catalytic core built around cullin scaffolds with ∼300 exchangeable substrate adaptors. To ensure robust signal transduction, cells must constantly form new CRLs by pairing substrate-bound adaptors with their cullins, but how this occurs at the right time and place is still poorly understood. Here, we show that formation of individual CRL complexes is a tightly regulated process. Using CUL3KLHL12 as a model, we found that its co-adaptor PEF1-ALG2 initiates CRL3 formation by releasing KLHL12 from an assembly inhibitor at the endoplasmic reticulum, before co-adaptor monoubiquitylation stabilizes the enzyme for substrate modification. As the co-adaptor also helps recruit substrates, its role in CRL assembly couples target recognition to ubiquitylation. We propose that regulators dedicated to specific CRLs, such as assembly inhibitors or co-adaptors, cooperate with target-agnostic adaptor exchange mechanisms to establish E3 ligase complexes that control metazoan development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Culina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Culina/genética , Células HEK293 , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
8.
Biochim Biophys Acta Gen Subj ; 1866(5): 130112, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35217128

RESUMO

BACKGROUND: N-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins. SCOPE OF REVIEW: This review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins. MAJOR CONCLUSIONS: Utilizing an efficient prokaryotic protein expression system and a new LC-MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases. GENERAL SIGNIFICANCE: The analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.


Assuntos
Manosiltransferases , Saccharomyces cerevisiae , Retículo Endoplasmático/metabolismo , Lipopolissacarídeos , Manosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
Ann Clin Lab Sci ; 52(1): 117-125, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35181625

RESUMO

OBJECTIVE: Previous research showed that ALG3 was associated with several cancers, but the function of ALG3 in bladder cancer (BC) was yet unknown. The purpose of this study was to investigate the relative expression of ALG3 in BC tissues and corresponding normal tissues and the relationship between the relative expression of ALG3 and clinical outcome in bladder cancer patients. METHODS: In this study, the expression of ALG3 in bladder cancer was detected by immunochemistry. In order to determine the cell proliferation and migration ability more accurately, we performed colony forming assay, MTT assay and wound healing migration assay. The role of ALG3 on tumor growth and metastasis was explored by animal model in vivo. RESULTS: ALG3 was expressed higher in bladder cancer than that in the normal tissues (P<0.05). At the same time, we found that there was a positive correlation between ALG3 expression and the prognosis (P<0.05). Moreover, we also discovered that the expression of ALG3 was associated with clinical pathological features (P<0.05). The proliferation and migration abilities of bladder cancer cell line T24 and 5637 were inhibited by silencing ALG3. In addition, the growth of bladder cancer cell line T24 cells were inhibited by silencing ALG3 in vivo. CONCLUSION: Silencing ALG3 plays a critical role in bladder cancer development and growth. It inhibits bladder cancer cells growth in vitro and in vivo.


Assuntos
Manosiltransferases , Neoplasias da Bexiga Urinária , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Manosiltransferases/metabolismo , Prognóstico , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
10.
APMIS ; 130(3): 181-192, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34978741

RESUMO

Mycobacterium tuberculosis (M. tuberculosis) Rv1002c encodes the protein O-mannosyltransferase (PMT), which catalyzes the transfer of mannose to serine or threonine residues of proteins. We explored the function of PMT in vitro and in vivo. Rv1002c protein was heterogeneously overexpressed in nonpathogenic Mycobacterium smegmatis (named as MS_Rv1002c). A series of trials including mass spectrometry, transmission electron microscope, biofilm formation and antibiotics susceptibility were performed to explore the function of PMT on bacterial survival in vitro. Mouse experiments were carried out to evaluate the virulence of PMT in vivo. PMT decreased the cell envelope permeability and promoted microbial biofilm formation. PMT enhanced the mycobacterial survival in vivo and inhibited the release of pro-inflammatory cytokines in serum. The function might be associated with an increased abundance of some mannoproteins in culture filtrate (CF). PMT is likely to be involved in mycobacterial survival both in vivo and in vitro due to increasing the mannoproteins abundance in CF.


Assuntos
Biofilmes/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/fisiologia , Manosiltransferases/metabolismo , Mycobacterium tuberculosis/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Permeabilidade , Virulência/fisiologia
11.
Int J Mol Sci ; 23(2)2022 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-35055170

RESUMO

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.


Assuntos
Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Adolescente , Adulto , Fatores Etários , Idoso , Líquido da Lavagem Broncoalveolar , Estruturas Celulares , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Masculino , Manosídeos/biossíntese , Manosídeos/genética , Manosiltransferases/biossíntese , Manosiltransferases/genética , Pessoa de Meia-Idade , Adulto Jovem
12.
Mol Microbiol ; 117(2): 450-461, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34875117

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins are found in all eukaryotes and are especially abundant on the surface of protozoan parasites such as Trypanosoma brucei. GPI-mannosyltransferase-I (GPI-MT-I) catalyzes the addition of the first of three mannoses that make up the glycan core of GPI. Mammalian and yeast GPI-MT-I consist of two essential subunits, the catalytic subunit PIG-M/Gpi14 and the accessory subunit PIG-X/Pbn1(mammals/yeast). T. brucei GPI-MT-I has been highlighted as a potential antitrypanosome drug target but has not been fully characterized. Here, we show that T. brucei GPI-MT-I also has two subunits, TbGPI14 and TbPBN1. Using TbGPI14 deletion, and TbPBN1 RNAi-mediated depletion, we show that both proteins are essential for the mannosyltransferase activity needed for GPI synthesis and surface expression of GPI-anchored proteins. In addition, using native PAGE and co-immunoprecipitation analyses, we demonstrate that TbGPI14 and TbPBN1 interact to form a higher-order complex. Finally, we show that yeast Gpi14 does not restore GPI-MT-I function in TbGPI14 knockout trypanosomes, consistent with previously demonstrated species specificity within GPI-MT-I subunit associations. The identification of an essential trypanosome GPI-MT-I subcomponent indicates wide conservation of the heterodimeric architecture unusual for a glycosyltransferase, leaving open the question of the role of the noncatalytic TbPBN1 subunit in GPI-MT-I function.


Assuntos
Trypanosoma brucei brucei , Animais , Glicosilfosfatidilinositóis , Mamíferos/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
13.
J Bacteriol ; 204(1): e0044721, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34633871

RESUMO

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.


Assuntos
Proteínas Arqueais/metabolismo , Dolicol Monofosfato Manose/metabolismo , Haloferax volcanii/enzimologia , Manosiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , Domínio Catalítico , Dolicol Monofosfato Manose/química , Etilenodiaminas , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Manosiltransferases/genética , Fenóis , Conformação Proteica , Domínios Proteicos
14.
Appl Microbiol Biotechnol ; 105(23): 8771-8781, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34738170

RESUMO

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive ß-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support ß-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.


Assuntos
Proteínas Ligadas por GPI/biossíntese , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Glicosilação , Manosiltransferases/genética , Manosiltransferases/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
15.
Microbiology (Reading) ; 167(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34676818

RESUMO

In Actinobacteria, protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and ß-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt ▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt ▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.


Assuntos
Manosiltransferases/química , Manosiltransferases/metabolismo , Streptomyces coelicolor/enzimologia , Alanina/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Membrana Celular/metabolismo , Sequência Conservada , Manosiltransferases/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Streptomyces coelicolor/virologia
16.
Am J Med Genet A ; 185(11): 3494-3501, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34467644

RESUMO

Congenital disorder of glycosylation type Ig (ALG12-CDG) is a rare inherited metabolic disease caused by a defect in alpha-mannosyltransferase 8, encoded by the ALG12 gene (22q13.33). To date, only 15 patients have been diagnosed with ALG12-CDG globally. Due to a newborn Slovak patient's clinical and biochemical abnormalities, the isoelectric focusing of transferrin was performed with observed significant hypoglycosylation typical of CDG I. Furthermore, analysis of neutral serum N-glycans by mass spectrometry revealed the accumulation of GlcNAc2Man5-7 and decreased levels of GlcNAc2Man8-9, which indicated impaired ALG12 enzymatic activity. Genetic analysis of the coding regions of the ALG12 gene of the patient revealed a novel homozygous substitution mutation c.1439T>C p.(Leu480Pro) within Exon 10. Furthermore, both of the patient's parents and his twin sister were asymptomatic heterozygous carriers of the variant. This comprehensive genomic and glycomic approach led to the confirmation of the ALG12 pathogenic variant responsible for the clinical manifestation of the disorder in the patient described.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Predisposição Genética para Doença , Manosiltransferases/genética , Polissacarídeos/genética , Defeitos Congênitos da Glicosilação/epidemiologia , Defeitos Congênitos da Glicosilação/patologia , Feminino , Testes Genéticos , Glicosilação , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , Polissacarídeos/metabolismo , Eslováquia/epidemiologia , Transferrina/genética
17.
Res Microbiol ; 172(7-8): 103874, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34492336

RESUMO

LppX is an important virulence factor essential for surface localization of phthiocerol dimycocerosates (DIM) in Mycobacterium tuberculosis. Based on Concanavalin A recognition, M. tuberculosis LppX (LppX-tb) was initially proposed to be glycosylated in M. tuberculosis and more recently this glycosylation was characterized by mass spectrometry analysis on LppX-tb expressed and purified from Corynebacterium glutamicum. Here, using this model organism and Mycobacterium smegmatis, we show that S16 and T18 residues of LppX-tb are indeed glycosylated with several hexoses units. Interestingly this glycosylation is strictly dependent on the mannosyl transferase PMT which, in M. tuberculosis, has been reported to be crucial for virulence. Using a site directed mutagenesis approach, we were able to show that the absence of S16 and T18 glycosylation does not alter phthiocerol dimycocerosates (DIM) localization in M. tuberculosis.


Assuntos
Proteínas de Bactérias/metabolismo , Lipídeos/análise , Lipoproteínas/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Glicosilação , Metabolismo dos Lipídeos , Lipoproteínas/química , Lipoproteínas/genética , Manosiltransferases/genética , Manosiltransferases/metabolismo , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Virulência , Fatores de Virulência/química , Fatores de Virulência/genética
18.
Rinsho Ketsueki ; 62(8): 944-953, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34497235

RESUMO

Paroxysmal Nocturnal hemoglobinuria, PNH is usually caused by the somatic mutation of X-linked PIGA gene followed by the clonal expansion of the GPI (glycosylphosphatidylinositol) anchor defective hematopoietic stem cell clone. There are two hypotheses for the mechanism of clonal expansion, one is selection theory, in which GPI deficient cells escape from attacks of cytotoxic cells, and another is benign tumor theory in which GPI deficient cells get the additional mutations and acquire proliferative nature. Recently, we identified two types of PNH patients caused by the biallelic mutation of PIGT on chromosome 20 and PIGB on chromosome 15. Both PNH clones had the germ-line mutation in one allele and another allele was somatically mutated in a hematopoietic stem cell. Both somatic mutations were loss of heterozygosity (LOH), deletion in PIGT-PNH and copy neutral LOH (CN-LOH) in PIGB-PNH. These PNH patients had typical PNH symptoms, but they have in addition auto-inflammatory features. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. Similarly, in PIGB-PNH cells, GPI intermediates are accumulated and expressed on the cell surface. Those GPIs together with complement activation cause the inflammasome activation.


Assuntos
Hemoglobinúria Paroxística , Alelos , Glicosilfosfatidilinositóis/genética , Células-Tronco Hematopoéticas , Hemoglobinúria Paroxística/genética , Humanos , Manosiltransferases , Mutação
19.
Fungal Biol ; 125(10): 776-784, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34537173

RESUMO

The mannosyltransferase Alg9 plays a vital role in N-linked protein glycosylation in Saccharomyces cerevisiae, but its function in most filamentous fungi is not clear. The present study characterized BbAlg9 (an ortholog of S. cerevisiae Alg9) in Beauveria bassiana to determine the roles of N-mannosyltransferase in biological control potential of the filamentous entomopathogenic fungus. The disruption of BbAlg9 led to slower fungal growth in media with various nutrition compositions. The conidiation of ΔBbAlg9 was less than that of the wild type from the third to the fifth day but showed no significant difference on the sixth day, suggesting that BbAlg9 affects the development of conidia rather than conidial yield of late stage. ΔBbAlg9 showed defects in conidial germination, multiple stress tolerances and the yield of blastospores, with altered size and density, and virulence in hosts infected via the immersion and injection methods. The deletion of BbAlg9 resulted in defects in cell wall integrity, including increased mannoprotein and glucan content and decreased chitin content, which were accompanied by transcriptional activation or suppression of genes related to cell wall component biosynthesis. Notably, deletion of the N-mannosyltransferase BbAlg9 altered the transcription levels of O-mannosyltransferase genes (Pmt and Ktr family). These data show that BbAlg9 is involved in the fungal development, conidial stress tolerance, cell wall integrity and virulence of B. bassiana.


Assuntos
Beauveria , Beauveria/genética , Parede Celular , Proteínas Fúngicas/genética , Deleção de Genes , Manosiltransferases/genética , Saccharomyces cerevisiae , Esporos Fúngicos , Estresse Fisiológico , Virulência
20.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 46(8): 915-919, 2021 Aug 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34565739

RESUMO

Alpha-dystroglycanopathy (α-DGP) is a group of congenital muscular dystrophy and limb band muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (α-DG). At present, there are few studies on the clinical manifestations, genetic characteristics, and diagnostic methods for α-DGP in China. Two cases of α-DGP caused by POMT1 and POMT2 gene mutations in the protein O-mannosyltransferases (PMTs) family were admitted to the Department of Pediatrics, Xiangya Hospital, Central South University. The 2 patients showed exercise retardation, with or without mental retardation. Serum level of creatine kinase (CK) was increased significantly. Electromyography showed myogenic impairment. Muscle biopsy was consistent with myopathy. Genetic test showed that both patients had compound heterozygous mutations, and the parents of the 2 patients were heterozygous with one of the mutations. There were c.824+1G>A, splicing and c.1777G>A, p.A593T in POMT1 gene, and c.604T>G, p.F202V and c.868C>T, p.P290S in POMT2 gene. The online database was used to predict the mutation sites and suggested the pathogenicity. Finally, one patient was diagnosed as congenital muscular dystrophy with mental retardation (CMD-MR) and the other was dystrophytype 2N (LGMD2N). PMTs family has similar sequences. Gene mutations can lead to different degrees of muscular dystrophy with the increase of serum level of CK. α-DG is easy to be misdiagnosed. Genetic examination is beneficial to early diagnosis, prognosis, and genetic counseling.


Assuntos
Deficiência Intelectual , Distrofias Musculares , Criança , Distroglicanas/genética , Heterozigoto , Humanos , Deficiência Intelectual/genética , Manosiltransferases , Distrofias Musculares/genética , Mutação
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