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Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymatic activity:
T Kanaitsuka1, R Bortell, L A Stevens
1Department of Medicine, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
Several proteins with NAD+:arginine ADP-ribosyltransferase (ART) activity are expressed in T cells and affect their function. Rat T cells that express the ART designated RT6 are determinants of the expression of autoimmune diabetes. In the mouse, a 35-kDa ecto-ART modulates the proliferation and functional activity of CTL. Here we report on mouse ARTs designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice. mRNAs for Rt6-1 and Rt6-2 were found in spleen, thymus, and intestinal tissue of both strains, but Rt6-1 mRNA in C57BL/6 mice was detected only at low levels. Rt6-1 and Rt6-2 cDNAs from both strains were cloned and sequenced. Predicted amino acid sequences of Rt6-2 were identical in both strains, but there was an in-frame stop codon in the sequence of Rt6-1 in C57BL/6 mice not present in BALB/c mice. Recombinant C57BL/6 Rt6-2 and BALB/c Rt6-1 proteins expressed in COS1 cells exhibited ART activity and were documented to be glycosylphosphatidylinositol-linked membrane proteins. COS-1 cells transfected with a C57BL/6 Rt6-1 cDNA construct expressed a truncated protein consistent in size with that predicted by the presence of the stop codon. This approximately 21-kDa protein appeared not to be glycosylphosphatidylinositol linked to the cell surface and lacked ART activity. C57BL/6 Rt6-1 therefore appears to be a naturally occurring ART knockout. The expression of Rt6-1 and Rt6-2 mRNAs in lymphoid tissues suggests that these ARTs may regulate immune system functions. Expression of Rt6-2 or another redundant ART may compensate for the lack of enzymatically active Rt6-1 in C57BL/6 mice.
Insights
Mouse T cells express NAD+:arginine ADP-ribosyltransferase (ART) proteins that regulate immune function. A naturally occurring knockout of the Rt6-1 ART in C57BL/6 mice suggests functional redundancy in immune regulation.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- T cells express NAD+:arginine ADP-ribosyltransferase (ART) proteins impacting T cell function.
- Specific ARTs, like RT6 in rats and a 35-kDa ecto-ART in mice, are implicated in autoimmune diseases and T cell proliferation.
- Mouse strains BALB/c and C57BL/6 express distinct forms of Rt6 ARTs.
Purpose of the Study:
- To characterize mouse NAD+:arginine ADP-ribosyltransferases (ARTs) designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice.
- To investigate the functional consequences of genetic variations in Rt6-1 between mouse strains.
- To explore the potential role of these ARTs in regulating immune system functions.
Main Methods:
- Cloning and sequencing of Rt6-1 and Rt6-2 cDNAs from BALB/c and C57BL/6 mice.
- Expression of recombinant proteins in COS1 cells to assess ART activity and membrane localization.
- Analysis of mRNA expression in various tissues including spleen, thymus, and intestinal tissue.
Main Results:
- Rt6-1 and Rt6-2 mRNAs were detected in spleen, thymus, and intestinal tissues, with lower Rt6-1 mRNA levels in C57BL/6 mice.
- A genetic variation, an in-frame stop codon, was identified in the Rt6-1 sequence of C57BL/6 mice, absent in BALB/c mice.
- Recombinant Rt6-2 and BALB/c Rt6-1 proteins showed ART activity and were GPI-linked, while C57BL/6 Rt6-1 produced a truncated, non-functional, non-GPI-linked protein, acting as a natural knockout.
Conclusions:
- C57BL/6 Rt6-1 functions as a naturally occurring NAD+:arginine ADP-ribosyltransferase (ART) knockout due to a premature stop codon.
- The expression of Rt6-1 and Rt6-2 in lymphoid tissues suggests a role in regulating immune system functions.
- The lack of functional Rt6-1 in C57BL/6 mice may be compensated by Rt6-2 or other redundant ARTs, highlighting potential immune regulatory pathways.