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Characterization of beta-amyloid precursor proteins with or without the protease-inhibitor domain using anti-peptide
1Institute of Bio-organic Chemistry, Syntex Research, Palo Alto, California 94304.
Abstract:
Alternative splicing of the transcript encoding the beta-amyloid precursor protein (BAPP) of Alzheimer's disease produces multiple mRNA species. Translation of these mRNAs predicts protein products of 770, 751, and 695 amino acids. The difference arises from the inclusion in BAPP-770/751 of a 56-residue insert region which is homologous to Kunitz-type protease inhibitors. We have prepared and affinity-purified anti-peptide antibodies that react specifically with either BAPP-770/751 (insert-specific) or BAPP-695 (junction-specific). A detectable level of the mRNA corresponding to the BAPP-770/751 protein was found in all cell lines tested. Immunoprecipitation of 35S-labeled proteins from these cell lines showed them to contain one or two Mr 105,000 bands reactive with the insert-specific serum, i-291. In contrast, only cos-7 cells and the human neuroblastoma cell line, IMR-32, contained mRNA species that encode the BAPP-695 protein, as shown by Northern analysis with a junction-spanning oligonucleotide probe. A band of Mr 95,000 was immunoprecipitated specifically from these two cell lines using the junction-specific serum, J-284. Indirect immunofluorescence labeling of cells corroborated these findings. All cells reacted with the insert-specific antibodies, i-291 and i-324. Only cos-7 and IMR-32 cells reacted with the junction-specific antibody, J-284. These results demonstrate the usefulness of anti-peptide antibodies for the differential detection of the BAPP-695 and BAPP-770/751 proteins.
Insights
Researchers developed specific antibodies to differentiate between beta-amyloid precursor protein (BAPP) variants. This advancement aids in detecting BAPP-695 and BAPP-770/751 proteins, crucial for Alzheimer's disease research.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Alternative splicing of beta-amyloid precursor protein (BAPP) transcripts generates diverse mRNA species.
- These mRNAs translate into BAPP protein isoforms of 770, 751, and 695 amino acids.
- The BAPP-770/751 isoforms include a 56-amino acid insert homologous to Kunitz-type protease inhibitors, distinguishing them from BAPP-695.
Purpose of the Study:
- To develop and validate specific anti-peptide antibodies for distinguishing between BAPP-695 and BAPP-770/751 protein variants.
- To investigate the differential expression of BAPP isoforms in various cell lines.
Main Methods:
- Preparation and affinity purification of anti-peptide antibodies: insert-specific (for BAPP-770/751) and junction-specific (for BAPP-695).
- Detection of mRNA species using Northern analysis with a junction-spanning oligonucleotide probe.
- Immunoprecipitation of 35S-labeled proteins and indirect immunofluorescence labeling to detect protein expression.
Main Results:
- All tested cell lines expressed mRNA for BAPP-770/751, with corresponding ~105 kDa proteins detected by insert-specific antibodies.
- Only cos-7 and IMR-32 cells expressed mRNA for BAPP-695, with a specific ~95 kDa protein detected by junction-specific antibodies.
- Immunofluorescence confirmed widespread reactivity with insert-specific antibodies and selective reactivity with junction-specific antibodies in cos-7 and IMR-32 cells.
Conclusions:
- Anti-peptide antibodies are effective tools for the differential detection of BAPP-695 and BAPP-770/751 proteins.
- The study highlights the differential expression patterns of BAPP isoforms across different cell types.
- These findings contribute to understanding BAPP heterogeneity in cellular contexts relevant to Alzheimer's disease.