Rao Guruprasad, Santhoshkumar Puttur, Sharma K Krishna
Department of Ophthalmology, University of Missouri-Columbia, Columbia, MO 65212, USA.
Mol Vis. 2008 Mar 26;14:666-74.
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.
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).
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.
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.
我们之前的研究在正常年轻人、正常老年人以及老年白内障患者晶状体的尿素可溶部分中鉴定出了23种低分子量（<3.5 kDa）的晶状体蛋白肽。我们发现，这些晶状体蛋白片段之一，即存在于老年和白内障晶状体中的βA3/A1(102 - 117)肽（SDAYHIERLMSFRPIC），增加了β-晶状体蛋白、γ-晶状体蛋白和乙醇脱氢酶（ADH）的光散射，并且还降低了αB-晶状体蛋白的伴侣样活性。本研究旨在鉴定βA3/A1(102 - 117)肽在αB-晶状体蛋白中的相互作用位点。
首先用磺基琥珀酰亚胺基-2-[6-(生物素酰胺基)-2-{对叠氮苯甲酰胺基}-己酰胺基]乙基-1,3-二硫代丙酸酯（磺基-SBED）对βA3/A1(102 - 117)肽进行衍生化，磺基-SBED是一种可光活化的、含有生物素的异三功能交联剂。然后将生物素衍生化的肽与αB-晶状体蛋白在37℃下孵育2小时以形成复合物，随后进行光解以促进生物素标签从肽转移到αB-晶状体蛋白上。通过蛋白质印迹法确认标签转移，并用胰蛋白酶消化标记的αB-晶状体蛋白。携带生物素标签的αB-晶状体蛋白的胰蛋白酶肽通过抗生物素蛋白亲和色谱法纯化，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和纳喷雾四极杆飞行时间质谱（QqTOF MS/MS）鉴定αB-晶状体蛋白中βA3/A1(102 - 117)肽的相互作用位点。
我们发现βA3/A1(102 - 117)肽与αB-晶状体蛋白区域（70）LEKDR（74）、（83）HFSPEELKVK（92）、（91）VKVLGDVIEVHGK（103）、（93）VLGDVIEVHGKHEER（107）和（121）KYR（123）相互作用，这些区域是α-晶状体蛋白结构域的一部分，并且先前已被证明是αB-晶状体蛋白中功能性伴侣位点的一部分。βA3/A1(102 - 117)肽还与αB-晶状体蛋白COOH末端延伸区域（150）KQVSGPER（157）、（164）EEKPAVTAAPK（174）和（164）EEKPAVTAAPKK（175）相互作用。当βA3/A1(102 - 117)肽的两个疏水残基被亲水残基取代时，得到的取代肽SDADHGERLMSFRPIC不具有抗伴侣特性。
本研究证实了在老年和白内障晶状体中发现的源自βA3/A1-晶状体蛋白的低分子量肽与αB-晶状体蛋白之间的相互作用。βA3/A1(102 - 117)肽与αB-晶状体蛋白的伴侣位点和COOH末端延伸区域的结合可能解释了其抗伴侣特性。
