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Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane
T Yamauchi1, M Higashiura, T Yagura
1Department of Biology, Faculty of Science, Kwansei Gakuin University, Nishinomiya, Japan.
Abstract:
Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.
Insights
A novel monoclonal antibody (mAb G3A5) targets the 138-kDa Golgi protein (p138 antigen) in human and monkey cells. This integral membrane glycoprotein is involved in Golgi apparatus structure and function.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- The Golgi apparatus is a critical organelle for protein modification and transport.
- Understanding the molecular composition of the Golgi is essential for elucidating its functions.
Purpose of the Study:
- To generate and characterize a novel monoclonal antibody (mAb G3A5) targeting a specific Golgi apparatus protein.
- To identify and analyze the molecular properties of the target antigen (p138).
Main Methods:
- Production of monoclonal antibody (mAb G3A5) using partially purified Golgi membranes from HeLa cells.
- Indirect immunofluorescence microscopy to determine antibody specificity and cellular localization.
- Western immunoblotting, protease protection assays, salt wash, and sucrose density gradient centrifugation to characterize the p138 antigen.
- Immunoaffinity chromatography for antigen purification and N-glycosidase F treatment to analyze glycosylation.
Main Results:
- mAb G3A5 specifically labeled the Golgi apparatus in human and monkey cells, but not in bovine or mouse cells.
- Treatment with nocodazole and BFA caused fragmentation and redistribution of Golgi staining, indicating dynamic changes.
- Western blot analysis identified a single 138-kDa polypeptide (p138 antigen) recognized by mAb G3A5.
- p138 is an integral membrane protein of the Golgi apparatus, confirmed by protease protection, salt wash, and flotation assays.
- p138 antigen is a glycoprotein containing asparagine-linked carbohydrates, as evidenced by decreased molecular mass after N-glycosidase F treatment.
Conclusions:
- A novel monoclonal antibody, mAb G3A5, has been developed that specifically recognizes a 138-kDa integral membrane glycoprotein of the Golgi apparatus.
- The p138 antigen is conserved in human and primate Golgi but not in rodent cells, suggesting species-specific roles.
- The characterization of p138 provides new tools for studying Golgi apparatus structure, dynamics, and function.