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P. Brabet, L. Guillou, T. J. Guignard, M. O. Péquignot, K. Chekroud, C. Joly, Y. Boublik, C. P. Hamel; Reconstitution of the Human Visual Cycle Multiprotein Network in Insect Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3517. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
During the regeneration of rhodopsin in the dark, the isomerohydrolase (IMH) process of vitamin A in the rod/retinal pigment epithelium (RPE) visual cycle is a limiting step, which may be regulated by various mechanisms, including light, gene expression, metabolic compounds, chaperones, signaling pathways. Our goal was to reconstitute the IMH multiprotein complex to test some of these regulatory mechanisms.
The Bac-to-Bac baculovirus expression system was used to produce recombinant baculovirus stocks for individual and/or co-expression of human cDNA encoding lecithin retinol acyl transferase (LRAT), RPE-specific isomerase (RPE65), cellular retinal binding protein (CRALBP), and the fatty acid transport protein 1 (FATP1) in Sf9 insect cells. Cell homogenates were incubated with all-trans retinol (atRol), all-trans retinyl palmitate (atRP), or 11-cis retinol (11cRol) and both retinoid binding and enzymatic activities were evaluated. Metabolic compounds were added throughout the incubation time with retinoid substrates to assess enzymatic regulations.
All four recombinant proteins were expressed at high levels and the system allows for the control of stoichiometric LRAT:RPE65 ratios in an attempt to reconstitute nearly native conditions. Substrates mobilization was verified by differential centrifugation of soluble and membrane fractions and showed known specificities of both RPE65 and CRALBP for atRP and 11cRol, respectively. LRAT was active towards both atRol and 11cRol to produce retinyl esters, showed a velocity of 1.2 nmol/min/mg proteins for atRol, was saturable and inhibited by high amount of atRol. Interestingly, atRP was found as a minor proportion of produced total retinyl esters. The addition of 5 mM ATP had no effect on LRAT but remarkably decreased the production of retinyl esters in the presence of RPE65, together with the absence of produced 11cRol even with various LRAT/RPE65 ratios.
Insect cells are suitable to partially reconstitute the IMH process since no 11cRol was produced from atRol within different multiprotein ratios. However, LRAT activity was in the range of endogenous activity in RPE, saturable and regulated by atRol. ATP did not influence the LRAT and isomerase activities but might have increased RPE65-dependent hydrolase activity. Investigations are underway to test for FATP1 acyl-CoA synthase activities and possible regulatory effect on the LRAT and hydrolase activities.
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