A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule

Conor J. Crawford, Maggie P. Wear, Daniel F.Q. Smith, Clotilde d'Errico, Scott A. McConnell, Arturo Casadevall, Stefan Oscarson

Research output: Contribution to journalArticlepeer-review

Abstract

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans. From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

Original languageEnglish (US)
Article numbere2016198118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number5
DOIs
StatePublished - 2021

Keywords

  • Chemical biology
  • Fungi
  • Glucuronoxylomannan
  • Glycans
  • Host-microbe interactions

ASJC Scopus subject areas

  • General

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