Abstract
The discovery of antibody-mediated catalysis was a breakthrough that showed antibody function is not limited to specific binding interactions, and that immunoglobulins (Igs) may also chemically transform their target antigens. Recently, so-called “natural catalytic antibodies” have been intimately linked with several pathologies, where they either protect the organism or contribute to the development of autoimmune abnormalities. Previously, we showed that myelin-reactive autoantibodies from patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE) exhibit the ability to recognize and hydrolyse distinct epitopes within myelin basic protein (MBP). Further, the antibody-mediated cleavage of encephalitogenic MBP peptide 81–103, flanked by two fluorescent proteins, can serve as a novel biomarker for MS. Here, we report the next generation of this biomarker, based on the antibody-mediated degradation of a novel chemically synthesized FRET substrate, comprising the fluorophore Cy5 and the quencher QXL680, interconnected by the MBP peptide 81–99: Cy5-MBP81–99-QXL680. This substrate is degraded upon incubation with either purified antibodies from MS patients but not healthy donors or purified antibodies and splenocytes from EAE but not from non-immunized mice. Data presented herein suggest the elaboration of potential specific, rapid, and sensitive diagnostic criteria of active progressive MS.
Highlights
B cells contribute to the immune response via presentation of antigens, release of various cytokines, secretion of antibodies and may have immunosuppression functions[1]
Peptide MBP81-99 was chemically synthesized with a C-terminal cysteine and N-terminal 6-heptynoic acid, conjugated with QXL680-maleimide and sulfo-Cy5-azide via the standard protocol of copper-catalysed azide-alkyne click chemistry (Fig. 1b)
Monitoring of the signal corresponding to Cy5 fluorescence revealed that Cy5-MBP81–99-QXL680 was hydrolysed at least four-fold more rapidly by serum antibodies from immunized SJL mice, compared with the non-immunized SJL mice (Fig. 1c)
Summary
B cells contribute to the immune response via presentation of antigens, release of various cytokines, secretion of antibodies and may have immunosuppression functions[1]. The major functions of antibodies, produced by B cells, include pathogen neutralization, antibody-mediated phagocytosis, antibody-dependent cellular cytotoxicity, and complement-mediated lysis of pathogens and infected cells[2] In addition to these functions, the catalytic activity of immunoglobulins is elevated during pre-B-cell acute lymphoid leukaemia, acute myeloid leucosis, acquired immune deficiency syndrome[3], infections[4,5], and, especially, autoimmune disorders[6,7,8,9]. Antibodies from approximately 80% of MS patients were able to degrade the encephalitogenic MBP peptide 81–10330; in contrast, this ability was present in antibodies from only 9% of patients with other neuronal disorders and was entirely absent in antibodies from healthy donors This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a fluorescent sensor for the catalytic activity of immunoglobulins in studies of MS. We report the generation of the fluorescent peptide-based sensor of myelin-reactive catalytic activity of immunoglobulins, utilizing site-specific chemical conjugation of the MBP peptide 81–99 with fluorophore Cy5 and quencher QXL680
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
