Related Experiment Videos
The amyloid percursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell
L Autilio-Gambetti1, A Morandi, M Tabaton
1Division of Neuropathology, Case Western Reserve University, Cleveland, OH 44106.
Abstract:
The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pI of 6.2, while APP-228 failed to focus in the pH range of 4.7-7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.
Insights
Alzheimer disease research reveals amyloid precursor protein (APP) exists in two modified forms, APP-130 and APP-228, in intact cells. These forms suggest extensive posttranslational modifications of the original APP gene product.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Alzheimer disease (AD), normal aging, and Down syndrome involve amyloid deposits primarily composed of the A4 polypeptide.
- The A4 polypeptide originates from a larger precursor protein (APP), a known transmembrane glycoprotein.
- The characteristics of APP within intact cells remain largely unknown.
Purpose of the Study:
- To investigate the characteristics of the amyloid precursor protein (APP) in intact human and nonhuman cell lines.
- To identify and characterize different forms of APP present in fibroblasts.
- To understand the cellular localization and modifications of APP.
Main Methods:
- Immunoreactivity analysis of fibroblasts using antisera against A4 and other APP domains.
- Immunoblotting and two-dimensional gel electrophoresis to identify and analyze APP polypeptides.
- Sequential cell extraction and immunofluorescence staining to determine APP localization and properties.
Main Results:
- Two distinct APP forms were identified: a 130 kDa polypeptide (APP-130) and a 228 kDa polypeptide (APP-228).
- APP-130 was recognized by all antisera, while APP-228 was specifically recognized by the A4 antiserum.
- APP-130 is associated with nonionic detergents at high ionic strength, whereas APP-228 is a cytosolic component. Immunofluorescence indicated intracellular and plasma membrane localization.
Conclusions:
- APP-130 and APP-228 represent two distinct forms of APP resulting from significant posttranslational modifications.
- These modifications likely occur on a smaller original gene product.
- Similar posttranslational modifications of APP are probable across different cell types.