Fig 1.
OvGM2AP protein sequences of the various nematodes were retrieved from WormBase and aligned using PROMALS3D (A). Ov = Onchocerca volvulus (OVOC1952), Ts = Trichinella pseudospiralis (T4C_8078.1) 16%, Ll = Loa loa (EN70_10408) 72%, Ce = Caenorhabditis elegans (C34E7.4) 65%. Sequence colour representation; red = predicted alpha helices, blue = predicted beta strands; Consensus Structure (ss) symbols: e = beta strand, h = alpha helix. Consensus amino acid symbols: conserved amino acids = bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H, W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; alcohol (S, T): o; polar residues (D, E, H, K, N, Q, R, S, T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q, R, W, Y): b; positively charged (K, R, H): +; negatively charged (D, E): -; charged (D, E, K, R, H): c. Bold residues in the consensus sequence represent greater than 80% consensus. Numbers in the first row represent a level of conservation above 4. Schematic representation of the structure features of OvGM2AP. Phylogenetic analysis of OvGM2AP orthologues (B) of closely related nematodes: Onchocerca volvulus (OVOC1952), O. ochengi (nOo.2.0.1.g11554) 99%, Dirofilaria immitis (nDi.2.2.2.g07380) 78%, Brugia malayi (Bm2577) 72%, Wuchereria bancrofti (J9F8Z0) 73%, Loa loa (EN70_10408) 72%, Ascaris suum (ASU_07674) 66%, Toxocara canis (TCNE_0001193501) 69%, Caenorhabditis elegans (C34E7.4) 65% and A. lumbricoides (ALUE_0002125701) 65%. The Jones-Taylor-Thornton model was used to estimate protein distances for neighbor joining. Bootstrap values are indicated on the nodes.
Fig 2.
Structural annotations of the GM2AP.
Representation of human GM2AP domain organization (A(i)). Predicted lipid binding domain and disulfide bond positions for OvGM2AP are indicated as well (A(ii)). Disulphide bonds were predicted using the DISULFIND online prediction tool. 3D structure of human GM2AP (PDB access code 1G13) (B).
Fig 3.
Expression profile of OvGM2AP.
Transcriptional expression of OvGM2AP and OvGAPDH was evaluated from L1, L2, L3, adult male (AM) and adult female (AF) parasite stages by RT-PCR (A). PCR products were run on 1% agarose gels. Primers were designed for the specific amplification of 165 bp sequences of OvGM2AP and 192 bp sequences of OvGAPDH. OvGAPDH was used as a normalization control. OvGM2AP was also amplified from genomic DNA (gDNA) of the various parasite stages as control. Arrow indicates 957 bp target amplicon from gDNA while arrow head indicates a possible non-specific PCR product of approximately 600 bp. Mr = Promega 1Kb DNA ladder, C = No Template Control. OvGM2AP was also detected in 16 h ESP by western blot analysis using anti-OvGM2AP peptide antibody as primary antibody (B). Arrows indicate signals of OvGM2AP indicating the 28.8 KDa protein and possible oligomeric forms at approximately 85 and 110 KDa. The recombinant OvGM2AP expressed in Sf21 cells was analyzed by western blot after purification from IMAC Ni-NTA resin with 500 mM Imidazole (C). The flow-through (FT), the two wash fractions (W1 and W2) and the eluate (E) were analyzed by electrophoresis on a coomassie blue stained polyacrylamide gel (iii). Recombinant OvGM2AP was detected using serum from a rabbit immunized against an OvGM2AP peptide (anti-OvGM2AP) or a rabbit pre-immune serum (PI) (i). Sf21 cells, un-infected (lane 1) or infected by the recombinant virus expressing OvGM2AP were harvested 24 or 48 hours (lanes 2 and 3) after infection. The corresponding lysates were analysed by western blot using a mouse anti-His monoclonal antibody (1D11) (ii). Total IgG responses to the purified recombinant protein was analysed (D) using the indicated serum types (ESC = European serum control, HES = Hypoendemic serum and OVS = O. volvulus serum) as primary antibodies by western blot. Arrow heads indicate positions of OvGM2AP around 37 kDa and possible oligomers of around 85 and 110 kDa. A 12% SDS-PAGE gel was prepared in every instance of gel preparation using either a 40% acrylamide stock (C) or a 30% acrylamide stock (B and D).
Fig 4.
PNGase F Digestion analysis of OvGM2AP.
OvGM2AP was subjected to PNGase F digestion and analyzed on Coomassie blue stained SDS-PAGE gel and on nitrocellulose membranes by western blot (A). Arrow represents PNGase on coomassie staining which could not be detected using anti-OvGM2AP peptide antibodies on western blot. Protein exist predominantly in glycosylated form in the undigested fraction with a relatively small part of it unglycosylated (arrow head). MS analysis of the corresponding peptides revealed the presence of a deamidation at asparagine 173 in the deglycosylated (digested) protein but not in the glycosylated (undigested) protein (B); red circles indicate point of deamidation.
Fig 5.
Analysis of humoral immune response to OvGM2AP.
Purified 8X His-fused OvGM2AP was used to coat microtiter plates. Plates were blocked and later incubated with serum from indicated individuals (OVS, HES, or ESC), followed by secondary antibody of either goat anti-human IgG peroxidase conjugate (a) or mouse anti-human IgG1-4 (b-e) which was followed with goat anti-mouse IgG, peroxidase conjugate as tertiary antibody. The plates were revealed using TMB. Optical density of stopped reactions was read at 450 nm and OD values were plotted against the different serum types. OVS = O. volvulus Serum (n = 30), HES = Hypo-endemic Serum (n = 14), ESC = European serum control (n = 03). A one-way ANOVA was used to compare groups. Error bars represent median with range. Dotted horizontal lines represent cut-off at mean + 3 SD of HES.
Table 1.
Table of ROC values for IgG and IgG subclass responses to OvGM2AP.
ROC values were calculated for IgG and IgG subclass. Other parameters such as standard error (SE), confidence interval (CI), and P-values were also calculated for the IgG and IgG subclass immune responses to OvGM2AP. AUC = Area under the ROC curve.
Table 2.
Diagnostic accuracy parameters for OvGM2AP.
Cut off values were obtained at mean + 3 standard deviations of the hypoendemic serum (HES). Sensitivity refers to the probability of getting a positive test result in subjects with the disease; Specificity is the probability of a negative test result in a subject without the disease; Positive predictive value defines the probability of having the disease of interest in a positive result; Negative predictive value is the probability of not having the disease in a subject with a negative test result; Likelihood ratio for positive test results indicates how much more likely the positive test result is to occur in subjects with the disease compared to those without the disease; Likelihood ratio for negative test results represents the ratio of the probability that a negative test result will occur in subjects with the disease to the probability that the same result will occur in subjects without the disease.
Fig 6.
ELISA-analysis of IgG responses of treated individuals to OvGM2AP.
Purified 8X His-fused OvGM2AP was used to coat microtiter plates. Plates were blocked and later incubated with the indicated categories of serum followed by secondary antibody (goat anti-human IgG peroxidase conjugate). The plates were revealed using TMB. Optical density of stopped reactions was read at 450 nm and OD values were plotted against ivermectin treatment rounds (a) or different the different serum types (b). a) IgG responses to OvGM2AP in patients (n = 40) with different rounds of ivermectin intake including groups of ivermectin naïve patients, 0 (n = 10), 1–6 (n = 10), 7–12 (n = 10) and >13 (n = 10) rounds of ivermectin administration. b) IgG responses to OvGM2AP in different patient groups. OVS = O. volvulus Serum (n = 30), Bandjoun = serum samples from Bandjoun (n = 30), an onchocerciasis hypo-endemic zone, HES = Hypoendemic Serum (n = 14), ESC = European serum control (n = 03). A one-way ANOVA was used to compare groups. Error bars represent median with range.
Fig 7.
Analysis of humoral immune response of related nematode sera to OvGM2AP.
Purified 8X His-fused OvGM2AP was used to coat microtiter plates. Plates were blocked and later incubated with serum from indicated individuals (OVS, HES, LLS, WBS, BMS, MPS, ALS or ESC), followed by incubation with goat anti-human IgG peroxidase conjugate as secondary antibody. The plates were revealed using TMB. Optical density of stopped reactions was read at 450 nm and OD values were plotted against the different serum types. OVS = O. volvulus Serum (n = 30), HES = Hypo-endemic Serum (n = 14), ESC = European serum control (n = 03), LLS = L. loa serum (n = 39), WBS = W. bancrofti serum (n = 06), BMS = B. malayi serum (n = 03), MPS = M. perstans serum (n = 06), ALS = A. lumbricoides serum (n = 06). A one-way ANOVA was used to compare groups. Error bars represent median with range. Dotted horizontal lines represent cut-off at mean + 3 SD of HES.
Fig 8.
Analysis of GM2AP activity of OvGM2AP.
The ability of OvGM2AP to hydrolyze GM2 to GM3 was assessed in a liposomal setting (A). Liposomes were incubated with the following accordingly to the numbers; 1 = human recombinant GM2AP (negative control), 2 = OvGM2AP (negative control), 3 = β-hexosaminidase A (negative control), 4 = β-hexosaminidase A and tauridesoxicolate (positive control), 5–7 = β-hexosaminidase A and human recombinant GM2AP (positive control), 8–10 = β-hexosaminidase A and OvGM2AP. Varying concentrations of OvGM2AP were incubated with recombinant Hex A and MUG or MUGS artificial substrate (B) and levels of degradation of the substrates was assessed using a fluorimeter.