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

Abstract

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH2)3SH (glycan G29SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

Serodiagnosis and therapeutic monitoring of New-World tegumentary leishmaniasis using synthetic type-2 glycoinositolphospholipid-based neoglycoproteins

“…The mercaptopropyl glycoside derivative G29 SH (Gal f β1,3Manα-3-mercaptopropyl) structure was based on the L. major type-2 GIPL-1 [ 16 , 40 ] ( Figure 1 A), and it was synthesized as recently described [ 32 ]. The purity of the disulfide forms of G29 SH ([G29 S ] 2 ) was ∼95%, as assessed by nuclear magnetic resonance spectroscopy NMR spectroscopy ( 1 H-NMR [400 MHz, D 2 O, 300 K] and 13 C-NMR [100 MHz, D 2 O, 300 K]) of the disulfide form of G29 SH ([G29 SH ] 2 ) [ 32 ].…”

“…The mercaptopropyl glycoside derivative G29 SH (Gal f β1,3Manα-3-mercaptopropyl) structure was based on the L. major type-2 GIPL-1 [ 16 , 40 ] ( Figure 1 A), and it was synthesized as recently described [ 32 ]. The purity of the disulfide forms of G29 SH ([G29 S ] 2 ) was ∼95%, as assessed by nuclear magnetic resonance spectroscopy NMR spectroscopy ( 1 H-NMR [400 MHz, D 2 O, 300 K] and 13 C-NMR [100 MHz, D 2 O, 300 K]) of the disulfide form of G29 SH ([G29 SH ] 2 ) [ 32 ]. Upon reduction of (G29 SH ) 2 with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), the ensuing G29 SH was conjugated to commercial maleimide-derivatized BSA (Imject™ Maleimide-Activated BSA, Thermo Fisher Scientific) to give rise to NGP29b (Gal f β1,3Manα-BSA), whose purity (∼99%) was evaluated by MALDI-TOF-MS ( Figure 1 B-D) [ 32 ].…”

“…The purity of the disulfide forms of G29 SH ([G29 S ] 2 ) was ∼95%, as assessed by nuclear magnetic resonance spectroscopy NMR spectroscopy ( 1 H-NMR [400 MHz, D 2 O, 300 K] and 13 C-NMR [100 MHz, D 2 O, 300 K]) of the disulfide form of G29 SH ([G29 SH ] 2 ) [ 32 ]. Upon reduction of (G29 SH ) 2 with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), the ensuing G29 SH was conjugated to commercial maleimide-derivatized BSA (Imject™ Maleimide-Activated BSA, Thermo Fisher Scientific) to give rise to NGP29b (Gal f β1,3Manα-BSA), whose purity (∼99%) was evaluated by MALDI-TOF-MS ( Figure 1 B-D) [ 32 ]. The mercaptopropyl glycoside derivatives G30 SH (Gal p α1,3Gal f β1,3Manα-3-mercaptopropyl) and G28 SH (Gal p 1,6Gal p α1,3Gal f βα-3-mercaptopropyl) structures were based on L. major type-2 GIPL-2 and -3, respectively [ 16 , 40 ] ( Figure 1 A), and were synthesized as previously described [ 24 ].…”

“…In the present work, we expand these findings to similarly produced synthetic L. major type-2 GIPL-based NGPs applied to the serological diagnosis of patients across the TL spectrum caused by L. braziliensis . We show that sera from TL patients in the active phase of the disease strongly react with Gal f β1,3Manα-BSA (NGP29b) [ 32 ], Gal p α1,3Gal f β1,3Manα-BSA (NGP30b), and Gal p 1,6Gal p α1,3Gal f β-BSA (NGP28b) [ 24 ], respectively comprising the native L. major type-2 GIPL-1, -2, and -3 glycotopes. However, NGP28b exhibits higher sensitivity and specificity than do NGP29b and NGP30b.…”

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease

“…In this approach, information about the antigenicity of synthetic homogeneous oligosaccharides is obtained, rather than relying on the cultivation of parasites and the isolation and study of their glycoconjugates. Similar reversed immunoglycomic approaches have recently led to the discovery of β-galactofuranose (β-Gal f )-based BMKs for CCD diagnosis [ 42 ], and α-Gal-containing BMKs for the diagnosis of Old-World and New-World (American) cutaneous or tegumentary leishmaniases [ 43 , 44 ].…”