Characterization of Noncovalent Interactions between Human Milk Oligosaccharides and Proteins

• Author / Creator

  Chen, Yajie

• This thesis focuses on the characterization of noncovalent interactions between human milk oligosaccharides (HMOs) and proteins using electrospray ionization mass spectrometry (ESI-MS) based assays. HMOs play critical roles in protecting infants from viral and bacterial infections through noncovalent interactions with lectins. An method based on catch-and-release (CaR) ESI-MS assay was developed using three human galectin proteins as model systems. The method used three dimensional information that was extracted from purified HMOs: molecular weight (MW), ion-mobility separation arrival time (IMS-AT), and collision induced dissociation (CID) fingerprints to identify the binders. The result showed this method is able to simultaneously screen mixtures of free HMOs for binding to lectins in vitro.
  The method then was extended to screening HMO binders from natural libraries that extracted from pooled human milk. The HMO binders to a C-terminal fragment of human galectin 3 (hGal-3C) from natural libraries (human milk fractions) were identified. The assay was able to identify at least 35 HMO structures presented in the fractions. After screening against hGal-3C, the assay discovered a total of 17 molecular weights as the binder, and 11 of them correspond to 21 HMO structures that were previously shown to be ligands for this lectin.
  Discovery of the HMO binders to viral lectins is often a key for understanding the defensive mechanisms of HMO. Here, we identified and analyzed the interactions between the rotavirus spike protein VP8* domain and HMOs using a direct ESI-MS assay. The results showed that the binding patterns between different human rotavirus strains varies. Overall, the interactions of VP8* to HMOs are weak (Ka<104 M-1). G10P[11] showed preference in binding to type 2 lactosamine (LacNAc) and α2-6 sialic acid; G1P[8] showed almost no binding to HMOs; G2P[4] has various binding affinities to different structures in a range of 103 M-1 <Ka <104 M-1 and seems
  ii
  to prefer structures with one or more fucoses. The results suggested that the defensive mechanisms of HMOs could be both structure-depended and strain-depended.

• Subjects / Keywords

  • noncovalent
  • HMO
  • interactions

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3WP9TP3K

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.