Selection of the Alternative Exon 1 from thecd5Gene Down-Regulates Membrane Level of the Protein in B Lymphocytes

Abstract

The human cd5 gene has two alternative exons 1: exon 1A (E1A) which encodes the full-length (FL) CD5 protein and exon 1B (E1B) which encodes a truncated (TR) isoform. The FL variant of CD5 protein is translocated to the plasma membrane, while its TR variant is retained in the cytoplasm. Because there is an inverse relationship between the levels of FL-CD5 and TR-CD5 in B cells, we have addressed the issue of how the selection of exon 1 is determined. In leukemic B cells, DNA methyltransferase (DNMT)1-induced methylation of E1B prevents its transcription. Furthermore, the level of mRNA for DNMT1 correlates inversely with that of mRNA for CD5-E1B. However, suppression of E1B transcription is incomplete, and some molecules of TR-CD5 continue to be synthesized. Bortezomid-induced inhibition of the proteasome establishes that these TR-CD5 molecules are cleared through the ubiquitin-proteasome pathway. Transfection of CD5 mutants into COS-1 cells locates the ubiquitin-binding site at the second destruction box of the extracellular region of CD5. Activation of the B cells by anti-IgM, Staphylococcus aureus Cowan I (SAC), or PMA up-regulates DNMT1, and thereby CD5-E1A mRNA at the expense of CD5-E1B mRNA. Aberrant synthesis of TR-CD5 is thus offset by balanced degradation of excessive protein. Dysregulation of these mechanisms reduces the expression level of membrane CD5, and thereby diminishes the threshold of the response by cells expressing CD5.

IL-6 Modulates CD5 Expression in B Cells from Patients with Lupus by Regulating DNA Methylation

“…Results generated from our previous studies indicated that the modulatory effects of CD5 protein on BCR signaling are attributed to the CD5 isoform which contains E1A, the previously documented exon 1. Introduction of cDNA for the recently identified CD5-E1B isoform into CD5 ϩ T and B cells reduced membrane expression of CD5 protein (17,19). Coexpression experiments have also shown that in the presence of CD5-E1B, CD5-E1A was not translocated to the membrane but associated with CD5-E1B in cytoplasmic aggregates (19).…”

“…Introduction of cDNA for the recently identified CD5-E1B isoform into CD5 ϩ T and B cells reduced membrane expression of CD5 protein (17,19). Coexpression experiments have also shown that in the presence of CD5-E1B, CD5-E1A was not translocated to the membrane but associated with CD5-E1B in cytoplasmic aggregates (19). The exact dynamics of the interaction between CD5-E1B and CD5-E1A remain unknown.…”

“…1A). However, membrane density of CD5 on B1 cells was lower in the patients ( Expression of CD5-E1B, which is retained in the cytoplasm, reduces membrane expression of CD5-E1A (17,19). This suggests that CD5-expressing B cells may, under certain physiological conditions (such as activation), preferentially up-regulate CD5-E1B relative to CD5-E1A.…”

“…Indeed, CD5-E1B is a fusion transcript from a new exon (4) that incorporates the 5Ј long terminal repeat (LTR) of a human endogenous retrovirus (HERV) sequence integrated at the time of old and new world monkeys divergence estimated at ϳ25-30 million years ago (18). We recently observed that transcription of mRNA for this isoform inversely correlates with the level of DNMT1 mRNA (19).…”

“…Undigested genomic DNA was used as positive control. The PCR primers were positioned upstream and downstream of the recognition site in the promoters of E1A and E1B of the cd5, cd19, cd70, Pax5, Syk, and HRES-1 genes (11,19,21,22). The PCR protocol included an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 1 min, and primer extension at 72°C for 1 min; PCR cycles were followed by final extension at 72°C for 10 min.…”