May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Recognition Mapping as a Tool for Characterizing Ocular Mucins
Author Affiliations & Notes
  • S. C. Baos
    University of Bristol, Bristol, United Kingdom
    H. H. Wills Physics Laboratory; and Academic Unit of Ophthalmology,
  • M. Berry
    University of Bristol, Bristol, United Kingdom
    Academic Unit of Ophthalmology,
  • T. McMaster
    University of Bristol, Bristol, United Kingdom
    H. H. Wills Physics Laboratory,
  • Footnotes
    Commercial Relationships  S.C. Baos, None; M. Berry, None; T. McMaster, None.
  • Footnotes
    Support  Leverhulme Trust
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4780. doi:https://doi.org/
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      S. C. Baos, M. Berry, T. McMaster; Recognition Mapping as a Tool for Characterizing Ocular Mucins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4780. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose:
 

Further to the topological description of individual mucin polymers, we are seeking insights into mucin structure and extracellular configuration by probing the spatial distribution of significant epitopes in the peptide core, or within the mucin glycosylation.

 
Methods:
 

Mucins were extracted from supernatants of IOBA NHC epithelia, and fractionated by buoyant density and hydrodynamic volume. Using flexible NHS-PEG 8 nm linkers, Atomic Force Microscopy (AFM) tips were functionalised with antibodies (Santa Cruz Biotechnology). These were 45M1 and C-20, against the C-terminus of the MUC5AC peptide core, anti-MUC16 (N-20) and anti-MUC1 (N-19), which bind to the N-termini of MUC16 and MUC1 peptide cores respectively, and also WGA as a sugar-specific lectin. Control tips were functionalised with antibody-blocking peptide conjugates, and blocking sugar was injected while imaging with WGA-functionalised cantilevers. AFM, employing a proprietary liquid cell (Veeco, Santa Barbara, USA), was used to produce simultaneous topographic and force-extension curves, or force-volume maps. These highlight the localization, number and specific energies of recognition bonds.

 
Results:
 

Using anti-MUC5AC (C-20) as a probe, 70 % of the force-distance curves displayed specific interactions and 5 % in the presence of blocking peptide. Most recognition events occurred 20 - 40 nm from the mica surface in a region rich in mucin and 80 -100 nm where covering was sparse. Force distributions overlapped in the two regions, centred on 30 - 40 pN. The largest forces were measured in the sparsely covered region only, but at low relative frequency, which may suggest the possibility of multiple antibody-epitope binding events.  

 
Conclusions:
 

Differences in force and rupture distances result from mucin-mucin proximity, which restricts the number of interacting antibodies.

 
Keywords: cornea: surface mucins • microscopy: confocal/tunneling • glycoconjugates/glycoproteins 
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