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Mast cell heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast
1Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada.
Abstract:
Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two-dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS-PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2-8; pI 9-9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule-associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (less than 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1-9) and, in general, they were more acidic (pI about 8.5-9) than those of PMC. Four major basic proteins (IB6-9) were all 24 kDa but were slightly different in isoelectric points. These and another 46-kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP-binding proteins in IMMC. In spite of remarkable differences between basic granule-associated proteins in PMC and basic proteins in IMMC, spots in the neutral-acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule-associated proteins with serine protease activity.
Insights
Rat mast cells from different locations show distinct protein profiles, particularly in their basic granule-associated proteins. This heterogeneity impacts their serine protease activity and function.
Area of Science:
- Immunology
- Cell Biology
- Proteomics
Background:
- Mast cells are crucial immune cells found in various tissues.
- Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) are distinct subtypes.
- Understanding mast cell heterogeneity is key to comprehending immune responses.
Purpose of the Study:
- To compare the protein constituents of rat PMC and IMMC.
- To identify differences in granule-associated proteins and serine proteases between mast cell subtypes.
Main Methods:
- Purification of PMC and IMMC from Nippostrongylus brasiliensis-infected rats.
- Two-dimensional gel electrophoresis (2D-PAGE) for protein separation.
- Silver staining and [3H]diisopropyl fluorophosphate (DFP) binding for protein analysis.
Main Results:
- PMC exhibited seven dominant basic proteins (PB2-8) and numerous neutral/acidic proteins.
- IMMC possessed nine basic proteins (IB1-9), generally more acidic than PMC.
- Specific serine proteases (RMCP I in PMC, RMCP II in IMMC) were identified and characterized by DFP binding and antibody reactivity.
- Significant differences were observed in basic granule-associated proteins, while neutral-acidic proteins showed similarities with quantitative variations.
Conclusions:
- Rat mast cell populations from peritoneal cavity and intestinal mucosa display marked heterogeneity in protein composition.
- Differences in basic granule-associated proteins, including serine proteases, contribute to functional specialization.
- This heterogeneity suggests distinct roles for PMC and IMMC in immune regulation and host defense.