Murangi T, Prakash P, Moreira BP, Basera W, Botha M, et al.
The Journal of allergy and clinical immunology •
IgE to galactose alpha-1,3 galactose (alpha-gal) causes alpha-gal syndrome (delayed anaphylaxis after ingestion of mammalian meat). Development of sensitization has been attributed to tick bites; however, the possible role of other parasites has not been well studied. Our aims were to assess the presence, relative abundances, and site of localization of alpha-gal-containing proteins in common ectoparasites and endoparasites endemic in an area of high prevalence of alpha-gal syndrome, as well as to investigate the ability of ascaris antigens to elicit a reaction in a humanized rat basophil in vitro sensitization model. Levels of total IgE, Ascaris-specific IgE, and alpha-gal IgE were measured in sera from patients with challenge-proven alpha-gal syndrome and from controls without allergy. The presence, concentration, and localization of alpha-gal in parasites were assessed by ELISA, Western blotting, and immunohistochemistry. The ability of Ascaris lumbricoides antigen to elicit IgE-dependent reactivity was demonstrated by using the RS-ATL8 basophil reporter system. Alpha-gal IgE level correlated with A lumbricoides-specific IgE level. Alpha-gal protein at 70 to 130 kDa was detected in A lumbricoides at concentrations higher than those found in Rhipicephalus evertsi and Amblyomma hebraeum ticks. Immunohistochemistry was used to localize alpha-gal in tick salivary acini and the helminth gut. Non-alpha-gal-containing A lumbricoides antigens activated RS-ATL8 basophils primed with serum from subjects with alpha-gal syndrome. We demonstrated the presence, relative abundances, and site of localization of alpha-gal-containing proteins in parasites. The activation of RS-ATL8 IgE reporter cells primed with serum from subjects with alpha-gal syndrome on exposure to non-alpha-gal-containing A lumbricoides proteins indicates a possible role of exposure to A lumbricoides in alpha-gal sensitization and clinical reactivity.
Lund J, Takahashi N, Nakagawa H, Goodall M, Bentley T, et al.
Molecular immunology •
Oligosaccharide profiles were obtained for chimeric mouse-human antibodies corresponding to each of the human IgG subclasses 1-4, and mouse IgG2b antibodies each expressed in the mouse J558L cell line. These antibodies have specificity for the NIP hapten and form a matched set of IgGs. An IgG4 chimeric antibody (B72.3) produced in the chinese hamster ovary (CHO-K1) cell line was also analysed for carbohydrate. Additionally aglycosylated mutants of this IgG4 (B72.3) and anti-NIP mouse IgG2b were analysed. The total lack of carbohydrate found in the aglycosylated site-directed mutants human chimeric IgG4 B72.3 (Asn 297-->Gln) and mouse IgG2b (Asn 297-->Ala) demonstrates that there are no N-glycosylation sites other than Asn 297. Therefore glycosylation profiles for all the IgGs analysed reflect carbohydrate attached to this site. Factors such as cell type (A), template direction by the IgG heavy chains (B) and culture conditions (C) are shown to influence IgG glycosylation profiles. (A) The anti-NIP IgG antibodies expressed by the J558L cell line may have one or two Gal (alpha 1-->3) Gal residues per oligosaccharide unit, indicative of the presence of (alpha 1-->3) galactosyl transferase in the J558L mouse cell line. (B) The galactosylation profiles obtained for the IgG heavy chains, in particular the preference for galactosylation of the Man (alpha 1-->6) arm rather than the Man (alpha 1-->3) arm, contrary to the beta-galactosyltransferase specificity, suggest that the polypeptide chain may act as a template to influence the extent of galactosylation and hence the proportions of each oligosaccharide incorporated. The IgG2 antibody does not display this galactosylation preference. (C) The extent of galactosylation appears to be influenced by the growth conditions, with the highest levels of galactosylation being found for IgG produced by cells grown in still cultures, rather than cells grown as ascites or in hollow fibre bioreactors. It is concluded that though the profile of glycosylation is controlled predominantly by the glycosylation activity of the cell in which the IgG is expressed, differences between the IgG heavy chain templates of the various subclasses and culture conditions can also influence glycosylation.
Lund J, Takahashi N, Hindley S, Tyler R, Goodall M, et al.
Human antibodies and hybridomas •
Oligosaccharide profiles for monoclonal chimeric mouse-human and mouse IgG2b antibodies produced in the mouse J558L cell line have been determined. These chimeric antibodies share the same mouse light chain and VH region and have a single, complex, oligosaccharide moiety attached to residue 297 of the heavy chain. Since the panel of chimeric antibodies included each of the human IgG subclasses and a set of IgG3 proteins having single residue differences, it was possible to evaluate the contributions of the protein template, the cell line, and the culture conditions to glycosylation. The study shows that the J558L cell line glycosylates human heavy chains with oligosaccharides characteristic of mouse immunoglobulins. This includes a galactose alpha 1-->3 galactose structure not found in human IgG and for which most humans have naturally occurring "anti-Gal" antibodies. The extent of galactosylation was very dependent on the culture conditions and was maximal for still culture. The findings have significance for the production of antibodies for in vivo administration.
