1235 biotinylated lectin malii vector laboratories  (Vector Laboratories)

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: Evaluation of the sialylation status of flagellin isolated from PA +Sia (A) The sialic acid specific – Sambucus nigra (SNA) lectin conjugated to agarose beads was used to isolate sialoglycoproteins from PA +Sia lysate (100 µg) by a lectin-based pulldown assay. Highly sialylated bovine salivary mucin (BSM) was used as a positive control. In the case of PA +Sia lysate, the lectin bound sialylated proteins were resolved in 10% SDS-PAGE and visualized by Coomassie Blue staining. Staining revealed several fine bands along with a prominent band near 47 kDa and two bands near 34, 37 kDa. The presence of similar bands in the BSM control indicated that these 34, 37 kDa bands come from the SNA-agarose beads. The band near 47 kDa was excised and processed for in gel trypsin digestion. The protein band was reduced, alkylated then digested by trypsin and subjected to MALDI TOF MS/MS. The generated spectra were searched against a eubacterial protein database by the MASCOT search engine utilizing the Basic Local Alignment Tool (BLAST) of ABI GPS Explorer software, version 3.6. Through peptide mass fingerprinting, the protein band was identified as flagellin. The MS/MS spectra with each tryptic fragments denoted by respective m/z values and the BLAST search results are shown. The peptide fragments within the flagellin sequence which were detected via MALDI TOF MS/MS have been marked with red colour. (B) The isolated, purified Fla +Sia protein (2 μg) resolved to a single band in a 10% SDS-PAGE gel. This single band was carefully excised and subjected to in gel trypsin digestion as before. The peptide digest was analyzed via ESI MS/MS mode by the LTQ Orbitrap mass spectrometer. The generated spectra was searched against the eubacterial protein database compiled from Uniprot using the Sequest HT Proteome Discoverer 1.4 software (Thermo Fisher Scientific) utilizing the MASCOT search engine. All peptide fragments among the flagellin sequence which have been identified in the tryptic digest have been denoted with red colour. (C) Purified Fla +Sia (25 μg) was passively rehydrated into pH 4–7 IPG strips and resolved via isoelectric focussing. The resolved strips were placed over the resolving gel in a10% SDS-PAGE gel and fixed using melted agarose. The protein sample was resolved in the second dimension through electrophoresis. The 2D gels were stained using Coomassie Blue to reveal that the single band protein had split into seven isoforms . (D) The sialic acid content of Fla +Sia /Fla -Sia (500 µg) were estimated and compared using the fluorimetric acetyl acetone method following Shukla et al (1982). Following the acetyl-acetone reaction, sialic acids released from flagellin proteins formed a fluorigenic product which were quantified via a fluorimeter (Ex-410nm, Em-510nm). The amount of sialic acids present in each sample was calculated by comparing with a prepared standard dilution of synthetic N-acetyl Neuraminic acid. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (E) Fla +Sia /Fla -Sia (5μg) resolved via SDS-PAGE was transferred onto PVDF blots via wet transfer method. These blots were probed with biotinylated sialic acid binding lectins – Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA II). The blots were washed and incubated with secondary avidin-HRP and the extent of lectin binding was determined by developing the blot using chemiluminescent substrates. The Ponceau S stained blots are provided to account for equal protein loading. Densitometric data from three independent experiments were used to prepare the bar diagram shown here. Representative blots from one experiment are shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (F) Fla +Sia was used to coat a 96 well ELISA plate. A panel of Siglec-Fc chimera recombinant proteins (Siglec-3,4,5,6,7,9 and 10-Fc) was diluted and incubated in these Fla +Sia coated wells. The wells were then washed and incubated with anti-human IgG Fc-specific peroxidase secondary antibodies. The extent of Siglec-Fc remaining bound to the Fla +Sia coated wells were detected using TMB substrate. The colour which was generated from the bound secondary antibodies was measured as absorbance at 450nm in a plate reader. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (G) As before, Fla +Sia /Fla -Sia (5μg) were resolved in SDS-PAGE, transferred to blots and probed using the same Siglec-Fc panel. The blots were washed and then incubated with secondary anti-human IgG Fc region specific peroxidase antibodies. The extent of Siglec binding was directly visualized by developing the blots in a chemidoc using a chemiluminescent substrate. As before, Ponceau S stained blots are supplied to account for equal loading. One representative blot from three independent experiments performed is shown here.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Isolation, Positive Control, SDS Page, Staining, Control, Tandem Mass Spectroscopy, Generated, Software, Peptide Mass Fingerprinting, Sequencing, Purification, Mass Spectrometry, Electrophoresis, Binding Assay, Incubation, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: (A) Purified Fla +Sia or desialylated Fla +Sia (ds-Fla +Sia ) (20 μg) were passively rehydrated into pH 3-10 IPG strips. The proteins were resolved by isoelectric focusing following the manufacturer’s instructions. These strips were stained by Coomassie blue and then destained before being dried. IPG strips were observed to detect any changes in protein resolution upon sialidase treatment. Representative strips selected from the two independent experiments performed are shown here. (B) As before, Fla +Sia /ds-Fla +Sia (5μg) were resolved in SDS-PAGE, transferred on to PVDF blots and probed with biotinylated sialic acid binding lectins – SNA, MAA II ((5 μg/mL). The blots were washed and then incubated with avidin-HRP. The extent of lectin binding was determined by developing the blots in a chemidoc using a chemiluminescent substrate. Additionally, the galactose specific lectin – peanut agglutinin (PNA) was also used (10 μg/mL). Upon sialidase treatment, the removal of sialic acid residues at the glycan termini exposes the underlying galactose residues. PNA recognizes and binds these exposed galactose residues indicating the successful removal of sialic acids. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) In a same set up, Fla +Sia /ds-Fla +Sia blots were probed with Siglec-9-Fc chimera proteins. The blots were washed and then incubated with anti-human IgG Fc specific antibodies. Any change in Siglec-9 interaction with flagellin was detected by developing the blot in a chemidoc. As previously described, Ponceau S stained blots are shown to account for equal protein loading across lanes. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Purification, Staining, SDS Page, Binding Assay, Incubation, Avidin-Biotin Assay