bgl2  (New England Biolabs)

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Post-translational modifications of Bgl2 and Scw4 in T, G and L pools. Glut—glutathionylation, P—phosphorylation. Modified amino acid residues are underlined and highlighted in bold.

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques: Modification

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Analysis of extracts from Saccharomyces cerevisiae cell walls obtained with 0.1 M Tris for 3.5 h at 30 °C—T, with 6 M GuHCl for 2 h at 30 °C—G ( A – C ) and with water at 100 °C after removal of lipid component—L ( C , D ). PAGE stained with Coomassie G-250 ( A ) or with silver nitrate staining ( C ) and Western blot stained with antibodies against Bgl2 ( B , D ).

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques: Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Western blot analysis of Saccharomyces cerevisiae cell lysates obtained from WT-OE, N202-OE, and N284-OE strains. Samples were incubated for 15 min with (+) or without (−) endoglycosydase H (EndoH). Bgl2 bands are denoted by arrowheads; (*) indicates unglycosylated Bgl2. Staining with antibodies against Bgl2.

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques: Western Blot, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Microscopy of structures formed in G pool extracted from Saccharomyces cerevisiae cell walls: TEM ( A – F ), immunofluorescence microscopy, staining with antibodies against Bgl2 ( G – I ). General view of jellyfish-like associates obtained from wt strain ( A , B , G , H ), fibrillar structure of their bodies ( D , E ). Control samples from bgl2Δ strain ( C , F , I ).

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques: Microscopy, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Structural model of Bgl2 molecule without ( A ) and with glutathione ( B – D ). N-terminal is highlighted in green, C-terminal is highlighted in cyan. G32-S42 loop is shown in magenta. The glutathione molecule located in the cavity of Bgl2 greatly changes the structure ( B ). The external location of glutathione molecule does not contribute to significant changes in Bgl2 structure ( C , D ). Possible conformations of glutathione resulting from molecular dynamics simulation are shown as molecular surface (gray area). Axial ( A – C ) and frontal ( D ) views of the cartoon.

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Immunofluorescence microscopy of Saccharomyces cerevisiae cells and cell walls stained with antibodies against Bgl2. Scale bars correspond to 2 µm. Cells ( A – C ). CW with ( D , E ) and without ( F ) EDC crosslinking and boiled in 3% SDS. CW before ( G – I ) and after ( J – K ) Tris extraction. Control samples from bgl2Δ strain ( C , I , L ).

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: The hypothetical scheme of Saccharomyces cerevisiae cell wall segment with a microcompartment. We suppose that PTM-free Bgl2 enters the cell wall as a part of L pool. Depending on the degree of association with proteins in L pool, Bgl2 is directed to T or G pool. Probably, Bgl2, acquiring one or another set of PTMs, can migrate between T and G pools. In these two pools, at least some of the molecules are closely located, therefore, we suppose that they form the microcompartment. The localization of L pool could not be revealed. It is possible that proteins of L pool are dispersed rather than compactly located in the cell wall. Glut—glutathionylation, MultiP and MonoP—multi- and monophosphorylation, respectively.

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning

doi: 10.3390/ijms21218304

Figure Lengend Snippet: Saccharomyces cerevisiae strains used in present research.

Article Snippet: Samples were stained with mouse primary polyclonal antibodies against Bgl2 (obtained as described in the ) and secondary polyclonal goat anti-mouse antibodies IgG labeled with Alexa-488 fluorophore (Invitrogen, Moscow, Russia) or with rabbit primary polyclonal antibodies against Gas1 and secondary polyclonal goat anti-rabbit antibodies IgG labeled with Alexa-647 fluorophore (Invitrogen, Moscow, Russia; Rostock, Germany).

Techniques:

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Alignment of deduced amino acid sequences of Pneumocystis Bgl2 to that of yeasts. Bgl2 sequences of Pneumocystis carinii, Pneumocystis murina, and Pneumocystis jirovecii were aligned with Bgl2 of Saccharomyces cerevisiae and Schizosaccharomyces pombe using Clustal W. Identical or similar amino acid residues are boxed. The predicted signal peptide is double lined above the sequence. The catalytic active sites are denoted by asterisks, and the sites required for glucanosyltransferase activity are indicated by daggers. The 7 repeats in P. carinii sequence are marked by dashed lines above the sequences. GenBank accession numbers for P. murina, P. carinii, P. jirovecii, S. pombe, and S. cerevisiae bgl2 complementary DNA sequences are XM_019757731, XM_018372126, XM_018373049, CAB16200, and CAA97313, respectively.

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Sequencing, Activity Assay

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Expression of Pneumocystis murina Bgl2 recombinant protein. A, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the bacterial extracts expressing recombinant protein showed a prominent Coomassie blue stained band of the expected size, ~33 kDa (indicated by the arrow, lane 2). This band was not seen when bacterial extracts of control vector with no insert were analyzed (lane 1).The bacterial extracts of the expressed protein showed immunoreactivity with this band when probed with anti-His tag antibody (lane 3), but no immunoreactivity was seen with the control vector (lane 4) when analyzed by immunoblotting. B, The COS-1 cell extracts expressing rBgl2 showed immunoreactivity by immunoblot with a ~33 kDa band when probed with anti-Bgl2 antibody (lane 1), whereas no immunoreactivity was observed in the cell extracts prepared from the control vector with no insert (lane 2).

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Plasmid Preparation, Western Blot

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Bgl2 has both β-1,3-glucanase and glucanosyltransferase activity. A, Immunofluorescence labeling of β-1,3-glucan in Pneumocystis murina organisms following Bgl2 digestion demonstrates a decrease in cyst-associated β-1,3-glucan. Partially purified P. murina organisms were treated with trypsin to expose cell wall–associated glucans, followed by treatment with COS-1 cell lysate expressing rBgl2 (top panel) or control lysate (vector with no insert, bottom panel), prior to immunofluorescence labeling of β-1,3-glucan with dectin-Fc (to detect cyst forms). Fluorescence indicates binding to β-1,3-glucan. There are fewer cysts in Bgl2 digested sample compared to the control sample. B, Glucanosyltransferase activity of P. murina rBgl2. Extracts from COS-1 cells transfected with bgl2 construct were incubated with reduced laminaripentoase (rG5) oligosaccharide and the products formed at different time points were detected by quadrupole time of flight liquid chromatography–mass spectrometry. Extracts from COS-1 cells transfected with a no-insert vector served as negative control. rG2, rG3, rG6, and rG7 are the reduced sugars while G2, G3, G4 are nonreduced forms. The x-axis represents the time points (in minutes) that samples were tested; y-axis shows the peak area. The increase in the formation of rG6 and rG7 in the presence of rBgl2 when compared to control indicates that the expressed protein has glucanosyltransferase activity.

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Immunofluorescence, Labeling, Purification, Expressing, Plasmid Preparation, Fluorescence, Binding Assay, Transfection, Construct, Incubation, Liquid Chromatography, Mass Spectrometry, Negative Control

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Immunoreactivity of anti-rBgl2 antibody. A, rBgl2 expressed in bacteria was purified using nickel-chelating resin and used to immunize mice. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the purified protein showed a band of ~33 kDa protein when stained with Coomassie blue (lane 1). This protein showed immunoreactivity to both anti-His tag antibody (lane 2) and mouse anti-Bgl2 antibody (lane 3) by immunoblot analysis. B, Protein extracts prepared from partially purified Pneumocystis murina organisms were subjected to immunoblot analysis using anti-Bgl2 antibody. An immunoreactive ~50-kDa band of expected size as indicated by the arrow was detected (lane 1). This immunoreactivity was blocked when the antibody was preincubated with recombinant protein (lane 2).

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Purification, Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Immunofluorescence analysis of Bgl2 expression by Pneumocystis murina. Heavily infected lung sections from a CD40L knockout mouse were co-labeled with anti-Bgl2, anti-Msg, and factor G. Anti-Bgl2 (green, A), anti-Msg (red, B), factor G (magenta, C), and merge (D). There is co-localization of Bgl2 with Msg but not factor G, demonstrating that expression is associated with trophic forms but not cysts. The images represent maximum projection z-stacks obtained using a confocal microscope at ×400 magnification with zoom functionality. Bar is 10 microns.

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Immunofluorescence, Expressing, Infection, Knock-Out, Labeling, Microscopy

Journal: The Journal of Infectious Diseases

Article Title: Characterization of Pneumocystis murina Bgl2, an Endo-β-1,3-Glucanase and Glucanosyltransferase

doi: 10.1093/infdis/jiz172

Figure Lengend Snippet: Expression of Bgl2 in caspofungin-treated mice. A, Reverse-transcription quantitative polymerase chain reaction (qPCR) analysis of bgl2 messenger RNA (mRNA) expression in Pneumocystis murina. RNA extracted from caspofungin-treated or untreated (control) mouse lung samples was reverse transcribed and bgl2 mRNA expression was analyzed by qPCR. Pneumocystis murina 18S ribosomal RNA was used as an endogenous control. The x-axis shows the drug treatment and the y-axis shows the fold change in bgl2 mRNA expression compared to control. The animals treated with caspofungin for 9 days showed an increase (~2.3-fold) in the expression of bgl2 mRNA compared to the control. Caspofungin treatment for 21 days resulted in an ~45% decrease in bgl2 mRNA expression compared to control. P values indicated above each pair were determined using Student t test. B, Immunofluorescence analysis of Bgl2 expression by P. murina. Infected lung sections from a CD40L knockout mouse treated with caspofungin for 21 days were co-labeled with anti-Bgl2, anti-Msg, and factor G. Anti-Bgl2 (green, A), anti-Msg (red, B), factor G (magenta, C), and merge (D). As seen in nontreated Pneumocystis-infected mouse lung, Bgl2 is associated with Msg but not factor G, demonstrating that expression is associated with trophic forms but not cysts. The images represent maximum projection z-stacks obtained using a confocal microscope at ×400 magnification with zoom functionality. Bar is 15 microns.

Article Snippet: For recombinant protein, blots were probed with horseradish peroxidase–conjugated anti-His tag antibody (Santa Cruz Biotechnology); for P. murina –derived proteins, blots were probed with anti-Bgl2 antibody followed by horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Infection, Knock-Out, Labeling, Microscopy