Anti-chaperone betaA3/A1(102-117) peptide interacting sites in human alphaB-crystallin.
OBJECTIVE
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, betaA3/A1(102-117) peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by beta- and gamma-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of alphaB-crystallin. The present study was performed to identify the interacting sites of the betaA3/A1(102-117) peptide in alphaB-crystallin.


METHODS
betaA3/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 alphaB-crystallin at 37 degrees C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to alphaB-crystallin. Label transfer was confirmed by western blot, and the labeled alphaB-crystallin was digested with trypsin. Tryptic peptides from alphaB-crystallin carrying the biotin label were purified by avidin affinity chromatography, and betaA3/A1(102-117) peptide interacting sites in alphaB-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 betaA3/A1(102-117) peptide interacted with alphaB-crystallin regions (70)LEKDR(74), (83)HFSPEELKVK(92), (91)VKVLGDVIEVHGK(103), (93)VLGDVIEVHGKHEER(107), and (121)KYR(123), which are part of the alpha-crystallin domain, and were previously shown to be part of the functional chaperone site in alphaB-crystallin. The betaA3/A1(102-117) peptide also interacted with regions at the COOH-terminal extension of alphaB-crystallin, (150)KQVSGPER(157), (164)EEKPAVTAAPK(174), and (164)EEKPAVTAAPKK(175). When two of the hydrophobic residues of betaA3/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 betaA3/A1-crystallin found in aged and cataract lenses and alphaB-crystallin. The binding of betaA3/A1(102-117) peptide to the chaperone site and the COOH-terminal extension of alphaB-crystallin may explain its anti-chaperone property.
Rao
Guruprasad
G
Department of Ophthalmology, University of Missouri-Columbia, Columbia, MO 65212, USA.
Santhoshkumar
Puttur
P
Sharma
K Krishna
KK
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism*
D018832:Molecular Chaperones; D010455:Peptides; D011994:Recombinant Proteins; D038203:alpha-Crystallin B Chain