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Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development
D P Romancino1, G Ghersi, G Montana
1Istituto di Biologia dello Sviluppo, C.N.R., Palermo, Italy.
Insights
Researchers identified two butanol-extracted proteins (bep1 and bep4) from sea urchin embryos. Antibodies against these proteins inhibited cell reaggregation, suggesting their role in embryonic cell interactions.
Area of Science:
- Marine Biology
- Developmental Biology
- Biochemistry
Background:
- Cell-cell interactions are crucial for embryonic development.
- Identifying specific proteins involved in these interactions is key to understanding developmental processes.
Purpose of the Study:
- To identify and characterize novel proteins involved in sea urchin embryonic cell reaggregation.
- To investigate the role of butanol-extracted proteins (bep1 and bep4) in early sea urchin development.
Main Methods:
- Extraction of antigens from Paracentrotus lividus embryos using 3% butanol.
- Expression of bep1 and bep4 cDNA fragments as MS2 polymerase fusion proteins (1C1 and 4A1) in E. coli.
- Generation of polyclonal antibodies against fusion proteins and Western blot analysis.
- Immunolocalization studies and inhibition of cell reaggregation assays using Fab fragments.
Main Results:
- Two 33 KDa proteins, recognized by antibodies against bep1 and bep4, were identified in sea urchin blastula stage embryos.
- Evidence suggests these antigens exist as dimers (66 KDa) under reducing and non-reducing conditions.
- Antigens were localized to the cell surface.
- Fab fragments of anti-bep1 and anti-bep4 antibodies inhibited the reaggregation of dissociated embryonic cells.
Conclusions:
- The identified bep1 and bep4 proteins are likely involved in mediating cell-cell interactions during sea urchin embryonic development.
- These proteins may play a significant role in the reaggregation process of embryonic cells.
Abstract:
We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.
