Narindrasorasak S, Altman R A, Gonzalez-DeWhitt P, Greenberg B D, Kisilevsky R
Department of Pathology, Queen's University, Ontario, Canada.
Lab Invest. 1995 Mar;72(3):272-82.
Extracellular matrix proteins (ECMPs) of the basement membrane type, such as the heparan sulfate proteoglycan perlecan, laminin, entactin, collagen IV, and fibronectin are present in and have been implicated in the genesis of amyloids. As in many forms of amyloid, perlecan, laminin, collagen IV, and fibronectin are present in Alzheimer deposits. We have previously demonstrated high-affinity interactions between Alzheimer amyloid precursor proteins (beta PP-695, -751, and -770), and perlecan or laminin. With a view to examining our hypothesis that beta PP:ECMP interactions are involved in Alzheimer's amyloidogenesis, additional studies have now been performed examining the interactions of the beta PPs with entactin, fibronectin, and collagen IV, the influence each of the ECMPs has on the binding of the others to beta PPs, and the effect of beta PPs on interactions among the various ECMPs.
A modified solid-phase enzyme-linked immunosorbent assay was used to assess the binding of the various ECMPs to the beta PPs. One element was immobilized on plastic, and another element, operationally defined as a ligand, was incubated in solution at various concentrations over the immobilized protein. To evaluate the effect of one ECMP on the binding of other ECMPs to beta PP, the beta PP was immobilized and the binding of the "ligand" ECMP was assessed in the presence of a single concentration of a second "competitor" ECMP. Similarly, in evaluating the effect of beta PPs on the binding of ECMPs to each other, one ECMP was immobilized and the binding of a second ECMP "ligand" was assessed in the presence of a fixed concentration of beta PP "competitor."
As in the case of perlecan and laminin, each of the ECMPs bound to the beta PPs with high affinity (Kd values in the nanomolar range). The binding of entactin to beta PPs was stimulated by collagen IV but was markedly inhibited by laminin, perlecan, and fibronectin. Conversely, the presence of entactin inhibited the binding of perlecan, laminin, and fibronectin to beta PPs. Moreover, the presence of beta PPs usually interfered with the binding of ECMPs to each other. Generally, in all binding assays, beta PP-751 and -770, behaved in similar ways, but beta PP-695, the brain-specific form, exhibited unique characteristics.
These binding data may reflect the normal interactions of beta PPs with ECMPs. However, the fact that beta PPs interfere with the normal interactions between ECMPs themselves, a process that spontaneously generates a basement membrane, suggests that aspects of ECMP:beta PP binding may be a pathologic part of the amyloidogenic process in Alzheimer's disease.
基底膜类型的细胞外基质蛋白（ECMPs），如硫酸乙酰肝素蛋白聚糖核心蛋白聚糖、层粘连蛋白、巢蛋白、IV型胶原和纤连蛋白，存在于淀粉样蛋白中并与淀粉样蛋白的形成有关。与许多形式的淀粉样蛋白一样，核心蛋白聚糖、层粘连蛋白、IV型胶原和纤连蛋白存在于阿尔茨海默病沉积物中。我们之前已经证明了阿尔茨海默病淀粉样前体蛋白（βPP-695、-751和-770）与核心蛋白聚糖或层粘连蛋白之间存在高亲和力相互作用。为了检验我们的假设，即βPP：ECMP相互作用参与阿尔茨海默病的淀粉样蛋白生成，现在已经进行了额外的研究，以检验βPP与巢蛋白、纤连蛋白和IV型胶原的相互作用，每种ECMP对其他ECMP与βPP结合的影响，以及βPP对各种ECMP之间相互作用的影响。
使用改良的固相酶联免疫吸附测定法评估各种ECMP与βPP的结合。将一种成分固定在塑料上，另一种成分（在操作上定义为配体）在溶液中以不同浓度与固定化蛋白一起孵育。为了评估一种ECMP对其他ECMP与βPP结合的影响，将βPP固定化，并在单一浓度的第二种“竞争”ECMP存在下评估“配体”ECMP的结合。同样，在评估βPP对ECMP相互结合的影响时，将一种ECMP固定化，并在固定浓度的βPP“竞争剂”存在下评估第二种ECMP“配体”的结合。
与核心蛋白聚糖和层粘连蛋白的情况一样，每种ECMP都以高亲和力与βPP结合（Kd值在纳摩尔范围内）。巢蛋白与βPP的结合受到IV型胶原的刺激，但受到层粘连蛋白、核心蛋白聚糖和纤连蛋白的显著抑制。相反，巢蛋白的存在抑制了核心蛋白聚糖、层粘连蛋白和纤连蛋白与βPP的结合。此外，βPP的存在通常会干扰ECMP之间的相互结合。一般来说，在所有结合测定中，βPP-751和-770的行为相似，但脑特异性形式的βPP-695表现出独特的特征。
这些结合数据可能反映了βPP与ECMP的正常相互作用。然而，βPP干扰ECMP自身之间正常相互作用的事实，即自发形成基底膜的过程，表明ECMP：βPP结合的某些方面可能是阿尔茨海默病淀粉样蛋白生成过程中的病理部分。
