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Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes
T G Goodman1, H L Chang, W J Esselman
1Department of Cell Biology, Upjohn Company, Kalamazoo, MI 49001.
Insights
Murine intestinal intraepithelial lymphocytes (IEL) express a unique high molecular weight CD45 protein. This CD45 isoform, distinct from B cell forms, arises from IEL-specific post-translational modifications, not alternative splicing.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- CD45 is a crucial protein tyrosine phosphatase involved in lymphocyte activation.
- Intraepithelial lymphocytes (IEL) are immune cells residing in the intestinal epithelium, playing a critical role in mucosal immunity.
- Understanding CD45 expression and modification in IEL is vital for comprehending intestinal immune responses.
Purpose of the Study:
- To characterize the CD45 molecule expressed by murine intestinal intraepithelial lymphocytes (IEL).
- To investigate the molecular basis for the unique CD45 isoform found in IEL.
- To determine if alternative splicing or post-translational modifications account for the observed CD45 heterogeneity.
Main Methods:
- Immunofluorescent staining of CD8+ IEL with anti-CD45 monoclonal antibodies.
- Immunoprecipitation of CD45 molecules from IEL and analysis of their molecular weight (m.w.).
- Polymerase chain reaction (PCR) for CD45 variable exon usage analysis and restriction enzyme analysis.
- Metabolic labeling and pulse-chase experiments to study protein synthesis and modification.
Main Results:
- A high molecular weight (m.w. 260,000) CD45 isoform was identified, unique to IEL and distinct from B220.
- This high m.w. CD45 isoform expressed the CT1 determinant, found on Thy-1- IEL.
- CD45 mRNA in IEL showed significant usage of single or zero variable exons, unlike B cells.
- Pulse-chase experiments indicated that the high m.w. CD45 form resulted from post-translational modifications, likely glycosylation.
Conclusions:
- The high molecular weight CD45 isoform in IEL is generated through IEL-specific post-translational modifications, such as glycosylation.
- Alternative splicing of CD45 variable exons does not account for the unique IEL CD45 isoform.
- These findings highlight specialized CD45 regulation in intestinal immune cells.
Abstract:
The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.