Lipofuscin Formation Catalyzed by the Milk Protein β-Lactoglobulin: Lysine Residues in Cycloretinal Synthesis

Abstract

Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein β-lactoglobulin (BLG) can promote the cyclodimerization of all-trans-retinal to cycloretinal both in vitro and in vivo. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal in vivo. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD.

Alginate Trisaccharide Binding Sites on the Surface of β-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation

“…The positions of these binding sites are distinctly different from those determined for the AOSs, although Y102 is shared. Notably, BLGA is reported to catalyze lipofuscin formation using K60 and K69 as well as K77 and K91 as catalytic residues . Also, none of these residues were found to interact with the AOSs, even though side chain assignments are needed to fully confirm this conclusion.…”

“…Notably, BLGA is reported to catalyze lipofuscin formation using K60 and K69 as well as K77 and K91 as catalytic residues. 38 Also, none of these residues were found to interact with the AOSs, even though side chain assignments are needed to fully confirm this conclusion. Charged patches on BLG were previously suggested to engage in interaction with anionic polysaccharides, 13,15,16,18,20 and binding regions for anionic polysaccharides were hypothesized to include residues A1−K14, V41−K60, T76− K83, and A132−R148.…”

Catabolism of the Lipofuscin Cycloretinal by MsP1

“…This culture was cooled to 30 °C and induced with 1 mM IPTG for 4 h. The cells were pelleted (7000 rpm, 10 min), resuspended in 25 mL of column buffer (50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, and 10% glycerol), and stored frozen at −80 °C. The MBP-tagged TEV protease was purified using the protocol that was used to purify the BLG fusion protein . The presence of the protein was confirmed by SDS-PAGE analysis.…”

“…While Schiff base formation is proposed to play a major role in the biosynthesis of both cycloretinal 1 (either protein–lysine-mediated or phosphatidylethanolamine-mediated) and A2E 2 (phosphatidylethanolamine-mediated) (Figure ), Schiff base formation is also involved in the visual cycle and could be difficult to target through drug design.…”

β-Lactoglobulin and Glycodelin: Two Sides of the Same Coin?

“…The protein is abundant and easily prepared so that it has served as a convenient test-bed for essentially every molecular technique from absorption spectroscopy (Townend et al, 1960) to X-ray crystallography (Crowfoot and Riley, 1938) and zetapotential measurement (Chen and Dickinson, 1995) and pretty much everything else in between. Its ready availability has also led to redox (Conway et al, 2013;Corrochano et al, 2018), enzymic (Li et al, 1990;Gowda et al, 2017Gowda et al, , 2018 and co-factor (Pérez et al, 1992) properties being ascribed though that is hardly surprising. What is not always clear, is whether any of these observations have any direct relevance to the physiological function.…”