Journal: The Journal of Biological Chemistry

Article Title: Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques *

doi: 10.1074/jbc.M111.299545

Figure Lengend Snippet: GPBP is present in amyloid plaques. Shown are immunohistochemical stainings of GPBP/CERT and SAP in midtemporal cortex of AD and demented and non-demented control cases (cryostat sections). A, top, control case 47 (Braak 1B) white matter (WM) immunostained with anti-GPBP/CERT 300–350 shows many ramified microglia (see inset) and to some extent endothelial lining of blood vessels, whereas in the gray matter (GM), amyloid plaque staining and intense GPBP staining around the plaque core, reminiscent of clustered microglia, and in addition cytoplasmic staining of neuronal cells are seen. Bottom, demented control case (Case-294) with cortical changes but not sufficient to be classified as AD (Braak 2B). GPBP/CERT 1–50 immunostaining is seen in Aβ plaques and microglia, especially microglia in the white matter (top right) and near the core of a classical plaque (see also the higher magnification inset). Similar to anti-GPBP/CERT 1–50, plaque and microglial staining was seen with anti-GPBP/CERT 300–350, but less pronounced. With anti-SAP, in addition to plaques, also some cytoplasmic neuronal staining was observed (two central panels). B, immunofluorescence staining was performed to further investigate co-localization of GPBP and SAP. Fibrillar Aβ deposits in an AD case (case 264) were visualized with thioflavin S, SAP with monoclonal SAP-14, followed by goat anti-mouse HRP and rhodamine tyramide and GPBP with anti-GPBP/CERT 1–50- and Cy5-labeled goat anti-rabbit. Although both SAP and GPBP immunoreactivity co-localize with thioflavin positivity, their exact distribution differs (black and white images showing complete overlap in localization of thioflavin and SAP but not of GPBP). GPBP seems to be present in small cells resembling microglia clustered around Aβ and SAP deposits (arrow and lower panel (magnification)) as well as in many neuronal cells (arrowheads). Partial overlap in localization of GPBP and SAP within Aβ plaques was more clearly seen, when in the separate channels the GPBP signal was visualized as red and thioflavin or SAP as green (insets). Scale bars, 100 μm. C, Western blotting with a mAb anti-Aβ revealed that immunoprecipitation of either GPBP or SAP from brain lysates from an AD mouse model (APPswe/PS1ΔE9), but not from wild type littermates, co-isolated Aβ, which suggests the presence of Aβ-SAP and Aβ-GPBP complexes in AD model mouse brain. Aβ, GPBP, and SAP were immunoprecipitated from brain homogenates of control and Alzheimer transgenic mice using mouse mAb anti-SAP, clone 4E8 (Sigma) or mAb GPBP clone 3A1-C1, mAb β amyloid clone 6E10 (Covance). immunoprecipitation with nonspecific immunoglobulins was performed for control. Results shown are representative of three experiments.

Article Snippet: For immunohistochemical staining, 5-μm cryosections were mounted on coated glass slides (Menzel Gläser Superfrost PLUS, Braunschweig, Germany) and fixed in acetone for 10 min. Next, sections were incubated overnight with primary antibodies, including rabbit anti-SAP (Dako), mouse monoclonal anti-Aβ (clone 6F/3D, Dako), affinity-purified rabbit antibody specific for residues 300–350 of human GPBP/CERT (Bethyl Laboratories), or polyclonal rabbit anti-GPBP/CERT epitope 1–50.

Techniques: Immunohistochemical staining, Staining, Immunostaining, Immunofluorescence, Labeling, Western Blot, Immunoprecipitation, Isolation, Transgenic Assay