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Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes
T F Tedder1, G McIntyre, S F Schlossman
1Division of Tumor Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Molecular Immunology
|December 1, 1988
Summary
The B1 molecule (CD20) exists in multiple forms on B-lymphocytes, differing mainly in phosphorylation. These forms share common peptides and are not glycosylated, with phosphorylation potentially regulated by external stimuli like PMA.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The B1 molecule, also known as CD20, is a phosphoprotein exclusively expressed on B-lymphocytes.
- Understanding the heterogeneity of B1 molecule expression is crucial for B-cell biology.
Purpose of the Study:
- To investigate the different molecular forms of the B1 molecule expressed by B-lymphocytes.
- To determine the post-translational modifications, such as glycosylation and phosphorylation, that contribute to B1 heterogeneity.
Main Methods:
- Surface iodination and immunoprecipitation of B1 from B-cell lines and lymphocytes.
- Limited proteinase digestion to compare peptide maps of different B1 species.
- Biosynthetic labeling with [35S]methionine to analyze protein synthesis.
- Endoglycosidase digestion and tunicamycin treatment to assess glycosylation.
- Phosphorylation analysis and stimulation with phorbol 12-myristate 13-acetate (PMA).
Main Results:
- Two predominant B1 species were identified: Mr 33,000 (75-80%) and Mr 35,000 (20-25%).
- Peptide mapping indicated that these forms share common peptides, suggesting they are related.
- Biosynthetic labeling suggested the Mr 35,000 species might encompass Mr 34,500 and 36,000 forms.
- Neither the Mr 33,000 nor the Mr 34,500-36,000 forms were glycosylated.
- Both forms were constitutively phosphorylated, with PMA significantly increasing phosphorylation and the amount of the Mr 34,500-36,000 form.
Conclusions:
- Multiple forms of the B1 molecule are expressed by B-lymphocytes.
- The observed heterogeneity in B1 molecular weight is likely due to differential phosphorylation.
- PMA stimulation influences B1 phosphorylation and protein levels, suggesting a regulatory mechanism.