Journal: bioRxiv
Article Title: Recognition of highly branched N-glycans of the porcine whipworm by the immune system
doi: 10.1101/2023.09.21.557549
Figure Lengend Snippet: (A) T. suis extract was subject to SDS-PAGE followed by Western blotting with Concanavalin A and Aleuria aurantia lectin before and after PNGase F treatment; Ponceau S staining indicates a shift in the molecular weight of the major protein upon PNGase F digestion. (B) Western blotting with three innate immune system proteins, Dectin-2, DC-SIGN and MGL showing partial loss of staining after PNGase F treatment. (C) Coomassie blue staining of SDS-PAGE indicating the excised band of 46 kDa, which was subject to trypsin and PNGase A digestion, prior to labelling of the released N-glycans with 2-aminopyridine and off-line RP-HPLC/MALDI-TOF MS analysis; the MS/MS-verified structures are annotated. (D) Online LC-MS analysis of the tryptic peptides of the 46 kDa band indicate the highest score to a homologue of Poly-Cysteine and Histidine-Tailed Metalloproteins (PCHTP, otherwise known as p43; theoretical mass including signal peptide of 46.7 kDa) found in other Trichinellid species with an overall coverage of 83%; peptides with between 10 and 120 peptide spectrum matches are indicated in bold and potential N-glycosylation sites are in red.
Article Snippet: The slides were rehydrated in TSM buffer (20 mM Tris-Cl, 150 mM sodium chloride, 2 mM calcium chloride and 2 mM magnesium chloride) before incubation with (i) 10 µg/ml biotinylated lectins ConA, AAL, LTL, GNA, WGA, WFA, RCA and SNA (VectorLabs in 1xTSM, 0.05% Tween-20 and 1% BSA, i.e. TSMBB) followed by 2 µg/ml anti-biotin antibody AF 488 conjugated (Invitrogen) in TSMBB; (ii) 2 µg/ml anti-LDNF (L6B8 in TSMBB) ( ) and anti-FLDNF (F2D2 in TSMBB) ( ) followed by anti-mouse IgG AF 647 conjugated (1:1000, Invitrogen) in TSMBB or 5 µg/ml anti-mannose antibody (100-4G11-A) ( ) followed by 2 µg/ml anti-mouse IgM AF 647 (Invitrogen); (iii) 5 µg/ml human CRP (biotechne) in TSMBB with additional 2 mM Ca 2+ followed serially by anti-CRP from mouse (biotechne) in TSMBB and 2 µg/ml anti-mouse IgG AF 647 in TSMBB; (iv) 10 µg/ml TEPC-15 (Sigma-Aldrich) followed by 2 µg/ml anti-mouse IgA FITC in TSMBB; (v) 5 µg/ml human DC-SIGN-Fc (R&D System), 5 µg/ml human MGL-Fc (R&D System) and 1 µg/ml human Dectin-2-Fc (Sino Biological) in TSMBB followed by 5 µg/ml anti-mouse IgG AF 488 (Invitrogen); (vi) 1:250 dilution of uninfected and infected pig sera (provided by Dr. Andrew Williams, University of Copenhagen) followed by anti-pig IgG or IgM antibody (1:500 diluted in TSMBB).
Techniques: SDS Page, Western Blot, Staining, Molecular Weight, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy
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