PMC

MALDI-TOF mass spectrum of the CPI-GC released by mild acid treatment of the TV-LPG, plotted over the range m/z 3,000–25,000

FACE Analysis of endo-β-galactosidase treated CPI-GC. The CPI-GC was treated with endo-β-galactosidase for 18 h and the released saccharides were purified by C18 Sep-Pak and derivatized with ANTS for FACE analysis. Lane 1—standard glucose oligomers; lane 2—CPI-GC digest

Schematic of T. vaginalis LPG showing the major breakdown products e.g. outer saccharide branch and ceramide phospho-inositol glycan core (CPI-GC) cleaved by mild TFA hydrolysis

FACE analysis and predicted structure of endo-β-galactosidase digest of T. vaginalis LPG. Lane 1—total digest; lane 8—standard glucose oligomers. Bands B and A (marked with arrows) from lane 1 were eluted and further digested with exoglycosidases shown in lanes 2–4 (band B) and 5–7 (band A)

a MALDI-TOF mass spectrum of the glycans released from TV LPG by endo-β-galactosidase. b PSD spectrum of the [M + Na]⁺ m/z 771 in the spectrum shown above. All fragments except some of the peak centered at m/z 588 contain sodium. This peak likely contains contributions from the B₃(Na) fragment at m/z 587 and a C₃(H) fragment at m/z 591. c MALDI-TOF mass spectrum of the glycans released from TV-LPG by endo-β -galactosidase, after subsequent treatment with hexosaminidase

Quadrupole orthogonal TOF CID MS/MS spectra obtained during a nanospray and b capLC-nanospray MS analysis of TV LPG-derived glycans with a [M + Na]⁺ m/z 738.8, b [M + Na]⁺ m/z 1,187.6. Putative structures of the [M + Na]⁺ ions are shown for these major components from T. vaginalis LPG that were analyzed after release by endo-β-galactosidase or mild acid hydrolysis, reduction and permethylation. The component whose spectrum is shown in a was generated by both enzymatic and acid release; the component in b appeared in only the acid-released fractions

Dose-dependent upregulation of IL-8 after 24 h stimulation of vaginal epithelial cells with intact T. vaginalis LPG and various LPG fractions in two-fold serial dilutions. Controls included the diluents PBS and PBS/ethanol (EtOH), T. foetus (TF) LPG CPI-GC, live T. vaginalis (four to ten parasite per epithelial cell) and recombinant human IL-1β. Bars represent the mean and SEM of duplicate cultures in one of two experiments performed with each set of fractions (a and b)

SORI-CID mass spectrum obtained for the [M + Na]₊ m/z 960 in the MALDI-FTMS spectrum of oligosaccharides that were released from TV LPG by mild acid hydrolysis (outer branch saccharide). Compositions are indicated as dH, deoxyhexose; N, N-acetylhexosamine; P, pentose. Ions marked with an apostrophe (’) are B- (or Z)- type ions, the rest are Y- (or C)-type ions; loss of 74 u most likely originates through elimination of CHOHCHCH₂OH from a nonreducing HexNAc (or Pent) terminus of the intact glycan or cleavage of an internal HexNAc residue that has 1,4-linkage, to form the ^3,5X_n-series. Low m/z ions are internal ions. All ions contain Na

Effects of LPG fractions on epithelial cell viability measured by MTT assay. Endocervical (a) and vaginal (b and c) epithelial cells were exposed for 24 h to intact LPG or various LPG fragments and CPI-GC obtained after treatment with periodate, mild acid (TFA) or CPI-PLC (a and b). The CPI-GC and saccharide fractions shown in c were obtained after endo-β-galactosidase treatment of the native CPIGC. Stock solutions of lyophilized lipid fractions were prepared in PBS/ethanol (EtOH) and of saccharide fractions in PBS. Recombinant human (rh) IL-1β served as positive control. Horizontal axes present two-fold serial dilutions of LPG and fractions at equivalent concentrations. Bars represent means and SEM from triplicate cultures in one of three independent experiments with each cell line

Activation of NF-κB and ERK1/2 in endocervical (a) and vaginal (b–c) epithelial cells. Endocervical epithelial cell line stably transfected with pNF-κB lucifearse reporter (a) was exposed to intact TV and TF LPG, equivalent dose of native TV CPI-GC fraction and live T. vaginalis (four to ten parasite per epithelial cell), and rhIL-1β (25 ng/mL) for 24 h. Bars represent the means and SEM of luciferase activity in relative luminescent units (RLU) measured in triplicate cultures in two experiments. Vaginal epithelial cells were assessed for pNF-κB ser536 b at 2 h, pMEK1/2 c at 30 min and pERK1/2 d at 1 h post stimulation with TV LPG or equivalent dose (60 µg/mL) of CPI-GC. Bars represent means + SD from triplicate cultures in two experiments