Reversed Immunoglycomics Identifies α-Galactosyl-Bearing Glycotopes Specific for Leishmania major Infection

Abstract

All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi . The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major , the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Gal p α1,3Gal f β and Gal p α1,3Gal f β1,3Man p α glycotopes as diagnostic biomarkers for L. major- caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica- caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies.

Specific Recognition of β-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients

“…The benzoylated thiogalactofuranoside donor 1 [45] was used to glycosylate allyl mannoside acceptor 2 [46] to produce disaccharide 3. The regioselectivity using this or a similar mannosyl acceptor had previously been shown [40,42]. Hydrolysis of the benzylidene protecting group and radical addition of thioacetic acid to the terminal alkene afforded thioester 4.…”

“…We hypothesized that a β-Galf -based glycotope could be identified and exploited as a new specific BMK for CD which, unlike previously discovered glycan-based BMKs for CD, is free of α-Gal, and therefore shows less cross-reactivity with NHS. To address this hypothesis, we took a reversed immunoglycomics approach [40] that began with the synthesis of Galf β1,3Manpα-CH 2 CH 2 CH 2 SH (G29 SH ) and Galf β1,3Manpα1,2-[Galf β1,3]Manpα-CH 2 CH 2 CH 2 SH (G32 SH ) (Figure 1B). These target compounds contain terminal disaccharide and tetrasaccharide partial structures of a TCT GPI-anchor (Figure 1A) [31,33], respectively, and a 3-thiopropyl moiety allowing for conjugation to maleimide-derivatized BSA.…”

“…These target compounds contain terminal disaccharide and tetrasaccharide partial structures of a TCT GPI-anchor (Figure 1A) [31,33], respectively, and a 3-thiopropyl moiety allowing for conjugation to maleimide-derivatized BSA. Derivatives of Galf β1,3Manpα [41,42] and Galf β1,3Manpα1,2-[Galf β1,3]Manpα [41], as well as the terminal trisaccharide of a Leishmania type-2 GIPL [40,42], and a Leishmania lipophosphoglycan (LPG) core heptasaccharyl myo-inositol [43], and a T. cruzi LPPG heptasaccharyl myo-inositol [44] have previously been synthesized. Our synthesis of disaccharide G29 SH and its conjugation to commercially available maleimide-activated BSA is illustrated in Scheme 1.…”

“…The disulfide was reduced with TCEP followed by conjugation to commercial maleimide-derivatized BSA to furnish NGP32b, as observed by MALDI-TOF-MS (see Supplementary Material). With the NGP29b and NGP32b in hand, IgG Ab responses using sera from CCD patients could now be measured by CL-ELISA, as previously described [40,[47][48][49]. First, to identify the most suitable CL-ELISA conditions, sera from 10 patients with CCD from With the NGP29b and NGP32b in hand, IgG Ab responses using sera from CCD patients could now be measured by CL-ELISA, as previously described [40,[47][48][49].…”

“…With the NGP29b and NGP32b in hand, IgG Ab responses using sera from CCD patients could now be measured by CL-ELISA, as previously described [40,[47][48][49]. First, to identify the most suitable CL-ELISA conditions, sera from 10 patients with CCD from With the NGP29b and NGP32b in hand, IgG Ab responses using sera from CCD patients could now be measured by CL-ELISA, as previously described [40,[47][48][49]. First, to identify the most suitable CL-ELISA conditions, sera from 10 patients with CCD from an endemic region (Venezuela) and 10 sera from healthy individuals from a nonendemic region (USA) were pooled.…”