| 1. |
- Garbe, Julia, et al.
(författare)
-
EndoE from Enterococcus faecalis Hydrolyzes the Glycans of the Biofilm Inhibiting Protein Lactoferrin and Mediates Growth.
- 2014
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:3
-
Tidskriftsartikel (refereegranskat)abstract
- Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.
|
|
| 2. |
- Hayes, Jerrard M, et al.
(författare)
-
Fc Gamma Receptor Glycosylation Modulates the Binding of IgG Glycoforms : A Requirement for Stable Antibody Interactions
- 2014
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:12, s. 5471-5485
-
Tidskriftsartikel (refereegranskat)abstract
- FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIaPhe158/Val158, and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIaPhe158/Val158 receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIaPhe158 was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIaVal158 remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.
|
|
