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Niklaus H Mueller, Mitchell E Keener, Steven Droho; Heparin sulfate mediates lens cell uptake of αB-crystallin fused to the gC cell penetration peptide. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5304.
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α-Crystallin proteins are thought to prevent protein aggregation in lens, while loss of it results in cataract. Previous studies identified a cell penetration peptide (CPP), gC, that mediates the uptake of additional α-crystallin which may delay the onset of cataract. The purpose of this study was to determine cell receptors for gC-tagged αB-crystallin (gC-αB).
The gC-αB protein along with four αB-crystallin fused mutant CPPs were purified from E. coli cultures. Proteins were Alexa-labeled and incubated with wildtype or heparan sulfate (HS) deficient Chinese hamster ovary (CHO) cells at 37oC for one hour to allow for protein binding and uptake. Following incubation, cells were imaged by confocal microscopy and quantified. To determine the effects of proteins uptake by lens cells, FHL-124 cells were incubated with Alexa-labeled proteins and quantify by flow cytometry.
We determine that HS is required for gC-αB uptake. Using HS deficient CHO cells we found that almost no gC-αB bound to these cells as compared to wildtype controls. Most mutant CPPs with conserved changes had minimal changes on protein uptake. However, substitution of conserved arginine residues with lysine resulted in significantly decreased in uptake confirming their importance. Analysis of wildtype and mutant gC-αB uptake by FHL-124 cells by flow cytometry indicated similar findings.
gC-αB requires HS for cell efficient cell uptake. While some amino acid substitutions are permissible in the CPP, replacement of arginine residues drastically reduces protein uptake.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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