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Structure-function analysis of vascular tethering molecules using atomic force microscope.

機(jī)譯：使用原子力顯微鏡分析血管束縛分子的結(jié)構(gòu)功能。

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By studying the structure-function relationship of vascular adhesion molecules, such as L-selectin, GPIb-VWF, and ADAMTS13, which are involved in regulation of leukocyte and platelet adhesion to the vascular wall, we quantify the effects of three specific point mutations on L-selectin on its interaction kinetics with 2-GSP-6 and 6-sulfo-sLex, we characterize the kinetics of GPIbalpha as it interacts with the VWF A1 domain, and we characterize the kinetics of ADAMTS13 as it interacts with the VWF A1A2A3 tri-domain and we characterize its cleavage effects on A1A2A3. The overall project goal is to study how mechanical force regulates the binding kinetics of these proteins.;Selectin-ligand interactions (bonds) mediate the way leukocytes roll on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Atomic force microscopy is used to compare the kinetics of wild-type L-selectin with the kinetics of three mutants of L-selectin interacting with 2-GSP-6; these mutants are a synthetic glycosulfopeptide modeled after the binding site of PSGL-1, and 6-sulfo-sLex, and a synthetic glycan prototypical of PNAd. Rather than first prolong (catch) and then shorten (slip) bond lifetimes, increasing force monotonically shortened the lifetimes of L-selectin MutI (A108H+H110A) and MutIA (A108H) bonds with 2-GSP-6. MutIB (H110A) exhibited an augmented catch bond. L-selectin also formed catch-slip transitional bonds with 6-sulfo-sLex. In sharp contrast, MutI, MutIA and MutIB had no effect on the bond lifetimes. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and PNAd.;Although catch bonds have been observed for selectins interacting with their ligands, it is still not clear whether other cell adhesion molecules also exhibit catch bond behavior. The interaction between glycoprotein Ib (GPIb) and the von Willebrand Factor (VWF) mediates platelet translocation at the vascular vessel damage sites, which plays a critical role in initiating platelet adhesion and thrombus formation. Similar to L-selectin-mediated tethering and rolling of leukocytes, translocation of platelets on VWF requires a shear threshold, suggesting a possible catch bond at work there. We characterized the kinetics of GPIbalpha interacting with the VWF A1 domain, confirming that the catch bond existed. Two type 2B VWD A1 mutants eliminated the catch bond and gave longer low force lifetimes. The prolonged lifetimes at low force resulted in more agglutination of platelets with A1 coated microspheres in flow. Three type 2M VWD A1 mutants showed shifted catch-slip transitional bonds that exhibited shorter lifetimes at low force but longer lifetimes at high force level. A2A3 domains affected the GPIbalpha-A1 catch bond quantitatively. Type III collagen's capturing of A1 or A1A2A3 also quantitatively shifted their bond lifetimes with GPIbalpha, indicating that A1 could have different conformational states.;During the process of hemostasis, the size of prothrombotic ULVWF affects the affinity of VWF to platelets bearing GPIbalpha on the membrane. Seven years ago, ADAMTS13 was identified and characterized as a multi-domain metalloprotease that can cleave at the Tyr1605-Met1606 bond of VWF, thus regulating the size of ULVWF. We studied how force regulated the binding and cleavage of ADAMTS13 on VWF. The full length ADAMTS13 molecule formed catch-slip transitional bonds with A1A2A3 while CUB domains (CUB1&2) only formed slip bonds, suggesting that shear force may play a role in facilitating the enzyme's binding to its substrate. By utilizing the analysis of two force drop events, we found the cleavage effects could only be observed after the catastrophic structural change of A1A2A3. The putative uncoupling of A1 from the A2 domain could only have 14nm contour length increment and would not favor cleavage before A2 unfolding. The putative unfolding of the A2 domain would have much longer contour length increment capacity, depending on how many ss-sheets would be pulled out of the A2 domain. Unfolding the A2 domain exposed the ADAMTS13 cleavage site and favored the cleavage. Two protocols using different stretching molecules (GPIbalpha and CR1) and A1A2A3 immobilization methods (physical adsorption and anti-His capturing A3) revealed that the cleavage effects diminished with increases in stretching force. Regardless of single bond kinetics, time-to-unfold exhibited catch bond behavior for both stretching protocols, suggesting that catch bonds could also be observed during the domain internal structural change. (Abstract shortened by UMI.)
機(jī)譯：通過研究參與調(diào)節(jié)白細(xì)胞和血小板粘附于血管壁的血管粘附分子（如L-選擇蛋白，GPIb-VWF和ADAMTS13）的結(jié)構(gòu)-功能關(guān)系，我們定量了三個(gè)特定點(diǎn)突變對(duì)L-選擇素與2-GSP-6和6-磺基-sLex的相互作用動(dòng)力學(xué)，我們表征了GPIbalpha與VWF A1域相互作用的動(dòng)力學(xué)，并表征了ADAMTS13與VWF A1A2A3 tri相互作用的動(dòng)力學(xué)-域，我們表征其對(duì)A1A2A3的切割作用。該項(xiàng)目的總體目標(biāo)是研究機(jī)械力如何調(diào)節(jié)這些蛋白質(zhì)的結(jié)合動(dòng)力學(xué)。選擇素-配體相互作用（鍵）介導(dǎo)白細(xì)胞在血管表面滾動(dòng)的方式。選擇素識(shí)別差異配體的分子基礎(chǔ)了解甚少。原子力顯微鏡用于比較野生型L-選擇素的動(dòng)力學(xué)和L-選擇素與2-GSP-6相互作用的三個(gè)突變體的動(dòng)力學(xué)。這些突變體是按照PSGL-1和6-sulfo-sLex的結(jié)合位點(diǎn)和PNAd的合成聚糖原型建模的合成糖基糖肽。與其先延長(zhǎng)（捕獲）然后縮短（滑動(dòng)）鍵的壽命，不如增加力單調(diào)縮短了2-GSP-6的L-選擇素MutI（A108H + H110A）和MutIA（A108H）鍵的壽命。 MutIB（H110A）表現(xiàn)出增強(qiáng)的捕獲鍵。 L-選擇蛋白還與6-磺基-sLex形成了滑移過渡鍵。與之形成鮮明對(duì)比的是，MutI，MutIA和MutIB對(duì)鍵的壽命沒有影響。這些結(jié)果區(qū)分了L-選擇素結(jié)合PSGL-1和PNAd的分子機(jī)制。盡管已經(jīng)觀察到選擇素與其配體相互作用的捕獲鍵，但尚不清楚其他細(xì)胞粘附分子是否也表現(xiàn)出捕獲鍵行為。糖蛋白Ib（GPIb）和von Willebrand因子（VWF）之間的相互作用介導(dǎo)了血管損傷部位的血小板移位，這在啟動(dòng)血小板粘附和血栓形成中起著關(guān)鍵作用。與L-選擇蛋白介導(dǎo)的白細(xì)胞束縛和滾動(dòng)相似，血小板在VWF上的移位需要一個(gè)剪切閾值，這表明那里可能存在捕獲鍵。我們表征了與VWF A1域相互作用的GPIbalpha的動(dòng)力學(xué)，證實(shí)了捕獲鍵存在。兩個(gè)2B型VWD A1型突變體消除了捕獲鍵，并賦予了更長(zhǎng)的低作用力壽命。在低作用力下延長(zhǎng)的壽命導(dǎo)致血小板與A1包被的微球在流動(dòng)中發(fā)生更多的凝集。三種2M VWD A1型突變體表現(xiàn)出移位的滑移過渡鍵，這些鍵在低作用力下的壽命較短，而在高作用力下的壽命較長(zhǎng)。 A2A3域定量影響GPIbalpha-A1捕獲鍵。 III型膠原蛋白對(duì)A1或A1A2A3的捕獲也定量地改變了它們與GPIbalpha的鍵合壽命，表明A1可能具有不同的構(gòu)象狀態(tài)。 。七年前，ADAMTS13被鑒定為多域金屬蛋白酶，可以在VWF的Tyr1605-Met1606鍵處裂解，從而調(diào)節(jié)ULVWF的大小。我們研究了力量如何調(diào)節(jié)VWF上ADAMTS13的結(jié)合和裂解。全長(zhǎng)ADAMTS13分子與A1A2A3形成了捕獲-滑動(dòng)過渡鍵，而CUB域（CUB1＆2）僅形成了滑動(dòng)鍵，表明剪切力可能在促進(jìn)酶與其底物的結(jié)合中發(fā)揮作用。通過對(duì)兩個(gè)力下降事件的分析，我們發(fā)現(xiàn)分裂效應(yīng)只能在A1A2A3發(fā)生災(zāi)難性的結(jié)構(gòu)變化后才能觀察到。假定的A1與A2結(jié)構(gòu)域的解偶聯(lián)僅具有14nm的輪廓長(zhǎng)度增量，并且在A2展開之前不支持切割。 A2域的推定展開將具有更長(zhǎng)的輪廓長(zhǎng)度增量容量，這取決于從A2域中拉出多少?gòu)坰s-sheets。展開的A2結(jié)構(gòu)域暴露了ADAMTS13切割位點(diǎn)，并且有利于切割。兩種使用不同拉伸分子（GPIbalpha和CR1）和A1A2A3固定方法（物理吸附和抗His捕獲A3）的方案表明，裂解作用隨拉伸力的增加而降低。無(wú)論單鍵動(dòng)力學(xué)如何，展開時(shí)間在兩種拉伸方案中均表現(xiàn)出捕獲鍵行為，這表明在域內(nèi)部結(jié)構(gòu)變化期間也可以觀察到捕獲鍵。 （摘要由UMI縮短。）

著錄項(xiàng)

  • 作者

    Wu, Tao.;

  • 作者單位

    Georgia Institute of Technology.;

  • 授予單位 Georgia Institute of Technology.;
  • 學(xué)科 Engineering Biomedical.;Engineering Mechanical.;Biophysics General.
  • 學(xué)位 Ph.D.
  • 年度 2008
  • 頁(yè)碼 194 p.
  • 總頁(yè)數(shù) 194
  • 原文格式 PDF
  • 正文語(yǔ)種 eng
  • 中圖分類
  • 關(guān)鍵詞

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