July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Defining the Binding Interaction between Contactin3 and Amyloid Precursor Protein
Author Affiliations & Notes
  • Xi Peng
    Molecular Biology and Genetics, Johns Hopkins University, Baltimore, Maryland, United States
  • Jeremy Nathans
    Molecular Biology and Genetics, Johns Hopkins University, Baltimore, Maryland, United States
    Howard Hughes Medical Institute, Chevy Chase, Maryland, United States
  • Footnotes
    Commercial Relationships   Xi Peng, None; Jeremy Nathans, Cerevance (C)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3049. doi:
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      Xi Peng, Jeremy Nathans; Defining the Binding Interaction between Contactin3 and Amyloid Precursor Protein. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3049.

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

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Abstract

Purpose : Contactins (CNTNs) and amyloid precursor protein (APP) are widely expressed in the developing retina and play a critical role in directing the processes of retinal neurons to their appropriate retinal and brain targets. A previous study found that the 4 fibronectin (Fn) domains of CNTN4 interact with the E1 domain of APP. However, the specific sites of interaction between CNTNs and APP, and their corresponding 3-dimensional structural relationship have not yet been addressed. The aim of our study is to identify the specific amino acids of CNTN3 and APP involved in their interaction, and the spatial relationship of that interaction.

Methods : To identify the relevant binding domains, CNTN3 derivatives with various Fn domains deleted were produced as secreted Fc fusion proteins and the APP E1 domain and copper-binding domain (CuBD; a subdomain of APP E1) were produced as secreted alkaline phosphatase (AP) fusion proteins. To localize specific binding sites, clustered and then individual alanine substitution mutations were constructed in surface residues of CNTN3-Fn(1-2)-Fc and APP(CuBD)-AP fusion proteins. The CNTN3-Fc proteins were immobilized in the wells of a Protein-G-coated 96-well plate, and incubated with APP-AP proteins. The bound APP-AP probe was quantified using a colorimetric assay. Finally, the locations of amino acids that contribute to binding were mapped on the 3-dimensional structures of CNTN3 and APP.

Results : (a) Most or all of the critical sites reside in Fn(2) of CNTN3 and the CuBD domain. (b) For CNTN3, V752A eliminated binding, and R714A, R760A, and R764A greatly reduced binding. For APP, H137A and E183A eliminated binding, and block substitutions at residues 131, 132, and 134, and at 139 and 140 greatly reduced binding. (c) As three of the critical CNTN3 substitutions involved arginine residues (714, 764, and 760) that are arrayed along a line, and as three of the critical APP substitutions involved glutamate and aspartate residues (131, 183, and 139) that are arrayed along a line of roughly similar length, the most parsimonious explanation for the mutagenesis data is that these two surfaces directly interact.

Conclusions : These results identify the amino acids that are critical for CNTN3 and APP binding, and they constrain the 3-dimentional relationship of the interaction. These findings will inform future analyses of the mechanisms of neurite development in the visual system.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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