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X.F. Wang, R.J. Linhardt, J. Balsamo, J. Lilien, M.M. Jablonski; Purification of the putative permissive glycan receptor in the retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3649.
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Purpose: We have previously shown that lactose and structurally related glycans (e.g., galactose, IPTG) support the assembly of nascent photoreceptor outer segments in the absence of the RPE. We have also shown that permissive glycan–mediated support of photoreceptor outer segment assembly demonstrates characteristics of a receptor–mediated phenomenon, strongly suggesting the existence of a permissive glycan receptor in the retina. The purpose of the present study was to purify and identify the putative glycan receptor. Methods:Multivalent non–metabolizable ligands [asialo–galactosylated biantennary (NA2), and triantennary (NA3) glycans] with terminal galactose residues were used. The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in: Niu–Twitty medium; Niu–Twitty medium containing 5x10–6 to 5X10–11 M of NA2 and NA3. Control retinas matured in vitro with an adherent RPE. Photoreceptor outer segment organization was graded. To identify the putative glycan receptor, RPE–deprived retinas were incubated in biotinylated–NA3 ligand for 3 hrs at 230C. Two control conditions were used: one set of control eyes was exposed to biotinylated–NA3 plus a molar excess of unlabeled IPTG as competitive permissive sugar to eliminate non–specific interactions of the NA3 with retinal proteins; and additional control retinas were exposed to culture media without any permissive sugar. Retinas were then harvested, total protein was extracted and applied to an avidin column (ProFound Pull–Down biotinylated Protein: Protein Interaction Kit) to pull out all molecules that are biotinylated along with any proteins with which they are bound with a high affinity. The eluted protein(s) was run on 1D SDS–PAGE gel. Results: NA2 and NA3 optimally support outer segment assembly at 5x10–9 and 5X10–10 M, respectively. A single protein band was eluted from the column to which retina samples exposed to biotinylated–NA3 were applied, but not from that exposed to biotinylated–NA3 plus competitive sugar or culture media only. Conclusion:Both NA2 and NA3 support photoreceptor outer segment assembly in the nanomolar concentration range, indicating a very high affinity of the putative receptor for these multivalent ligands. Using biotinylated–NA3, we were able to isolate a single protein band from Xenopus retinas that was found in neither control condition. These data suggest that the isolated protein is the putative glycan receptor. A molecular characterization of the protein is pending.
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