Anti-chaperone βA3/A1(102-117) peptide interacting sites in human αB-crystallin

PURPOSE: Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, βA3/A1(102–117) peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by β- and γ-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of αB-crystallin. The present study was performed to identify the interacting sites of the βA3/A1(102–117) peptide in αB-crystallin. METHODS: βA3/A1(102–117) peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1–3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotin-containing cross-linker. The biotin-derivatized peptide was then incubated with αB-crystallin at 37 °C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to αB-crystallin. Label transfer was confirmed by western blot, and the labeled αB-crystallin was digested with trypsin. Tryptic peptides from αB-crystallin carrying the biotin label were purified by avidin affinity chromatography, and βA3/A1(102–117) peptide interacting sites in αB-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS). RESULTS: We found that the βA3/A1(102–117) peptide interacted with αB-crystallin regions (70)LEKDR(74), (83)HFSPEELKVK(92), (91)VKVLGDVIEVHGK(103), (93)VLGDVIEVHGKHEER(107), and (121)KYR(123), which are part of the α-crystallin domain, and were previously shown to be part of the functional chaperone site in αB-crystallin. The βA3/A1(102–117) peptide also interacted with regions at the COOH-terminal extension of αB-crystallin, (150)KQVSGPER(157), (164)EEKPAVTAAPK(174), and (164)EEKPAVTAAPKK(175). When two of the hydrophobic residues of βA3/A1(102–117) peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property. CONCLUSIONS: This study confirmed the interactions between a low molecular weight peptide derived from βA3/A1-crystallin found in aged and cataract lenses and αB-crystallin. The binding of βA3/A1(102–117) peptide to the chaperone site and the COOH-terminal extension of αB-crystallin may explain its anti-chaperone property.