Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

Abstract

We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT–Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate:protein ratios of 1.9:1,3.0:1,4.0:1,4.9:1, 5.9:1, 6.9:1, 7.9:1 and 9.1:1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate–protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT–Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein.

Defined Conjugation of Glycans to the Lysines of CRM197 Guided by their Reactivity Mapping

“…Determining the relative reactivity of lysine residues on the protein enables reaction conditions to be optimised, thereby directing glycoconjugation to the more‐accessible residues in order to obtain more homogenous products than those by conventional conjugation protocols. The finding that carbohydrate conjugation occurs at the more surface‐exposed sites is in agreement with a similar recent observation on neoglycoconjugates prepared by coupling carbohydrate antigens bearing a squarate linker to rTT‐Hc 16. However, in comparison to other reported methods, 12, 14–16] our approach presents two main advantages: firstly, the conjugation sites can be predetermined by rapid LC‐MS/MS analysis of the linker‐labelled protein digests, before carrying out the conjugation of precious synthetic glycans; and, secondly, by semiquantitative determination of the labelling level for the modified sites, the more reactive lysine residues can be identified and targeted for defined conjugation.…”

“…The same group has also used MALDI‐TOF/TOF‐MS to analyse digests from BSA to which of synthetic carbohydrate haptens from Vibrio cholerae (at various ratios) had been attached:15 only three of the 60 lysine residues were mapped. In follow‐up studies, LC‐MS/MS analysis of tryptic and GluC V8 digests enabled the identification of 12 glycation sites in neo‐glycoconjugates prepared by coupling an average of 5.5 V. cholerae haptens to recombinant tetanus toxin C‐fragment (rTT‐Hc), with a protein coverage of 83.6 % 16. It is interesting to note that although both BSA and rTT‐Hc are interesting models, neither is used in vaccines currently in clinical practice.…”

“…Following covalent linkage of glycans, MS analysis of the products is very laborious and challenging, even when well‐defined oligosaccharides are employed. Determination of the glycation sites is further complicated by possible decomposition of the carbohydrate antigens during mass analysis14–16 or by an increase in the length of the conjugated oligosaccharide.…”

Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys‐34) of BSA. Mapping of the glycation sites of the anti‐tumor Thomsen–Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction

“…For the determination of the average number of carbohydrate‐spacer moieties linked to BSA (TF antigen:BSA), the two glycoconjugates were analyzed by matrix assisted laser desorption/ionization‐mass spectrometry (Fig. a and b and Table ) . The MALDI‐TOF‐MS was characterized by the formation of a protonated molecular ions [M + H] + at m/z 67 599 and 70 905 and their TF–BSA ratios were calculated to be individually 2:1 and 8:1.…”

“…BSA is one of the most widely studied proteins used as a carrier protein for the synthesis of neoglycoconjugates. [27,[37][38][39][40][41] BSA contains 35 cysteine residues linked by S-S disulphide bonds among which, however, there is only a single buried free cysteine residue (Cys34) [42] that has been systematically used for other conjugation. [43,44] It is known that the 35 cysteine residues of BSA form 17 disulphide bonds at these positions (1) (17) 581-590 .…”