Abstract
Background and aims: The H2-A(g7)(Ag7) MHC class II (MHCII) allele is required for type 1 diabetes (T1D) in NOD mice. Ag7not only has a unique peptide binding profile, but has been reported to exhibit biochemical defects, including accelerated protein turnover. Such defects have been proposed to impair antigen presentation, and thus self tolerance. Here, we report the first measurements of murine MHCII protein synthesis and turnover in vivo.
Methods: NOD mice and BALB/c controls were labeled continuously with heavy water (2H2O), and splenic B cells and DCs were isolated. MHCII molecules were immunoprecipitated and digested with trypsin. Digests were analysed by liquid chromatography/mass spectrometry (LC/MS) to quantify the fraction of newly synthesized MHCII molecules, and thus turnover.
Results: MHCII turnover was faster in DCs than B cells, varying
slightly between mouse strains. Some Ag7 molecules exhibited accelerated turnover in B cells from young, but not older, prediabetic female NOD mice. This acceleration was not detected in a second NOD colony with a high incidence of T1D. Turnover rates of Ag7 and Ad were indistinguishable in (NOD x BALB/c) F1 mice.
Conclusions: Accelerated MHCII turnover may occur in NOD mice, but reflects environmental and developmental regulation, rather than a structural deficit of the Ag7 allele. Moreover, this phenotype wanes before onset of overt T1D and is dispensable for development of autoimmune diabetes. Our observations highlight the importance of in vivo studies in understanding the role of protein turnover in
genotype/phenotype relationships and offer a novel approach for addressing this fundamental research challenge.
Methods: NOD mice and BALB/c controls were labeled continuously with heavy water (2H2O), and splenic B cells and DCs were isolated. MHCII molecules were immunoprecipitated and digested with trypsin. Digests were analysed by liquid chromatography/mass spectrometry (LC/MS) to quantify the fraction of newly synthesized MHCII molecules, and thus turnover.
Results: MHCII turnover was faster in DCs than B cells, varying
slightly between mouse strains. Some Ag7 molecules exhibited accelerated turnover in B cells from young, but not older, prediabetic female NOD mice. This acceleration was not detected in a second NOD colony with a high incidence of T1D. Turnover rates of Ag7 and Ad were indistinguishable in (NOD x BALB/c) F1 mice.
Conclusions: Accelerated MHCII turnover may occur in NOD mice, but reflects environmental and developmental regulation, rather than a structural deficit of the Ag7 allele. Moreover, this phenotype wanes before onset of overt T1D and is dispensable for development of autoimmune diabetes. Our observations highlight the importance of in vivo studies in understanding the role of protein turnover in
genotype/phenotype relationships and offer a novel approach for addressing this fundamental research challenge.
| Original language | English |
|---|---|
| Pages (from-to) | 49 |
| Journal | IMMUNOLOGY |
| Volume | 140 |
| Issue number | s1 |
| Publication status | Published - 2013 |