Abstract
Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to Nε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.
Highlights
The covalent modification of proteins has been implicated in a variety of diseases, including atherosclerosis [1, 2]
Based on our previous finding that the apolipoprotein E (apoE) deficiency leads to a significant accumulation of pyrrolated proteins (pyrP) in mice [15], we first examined changes in the serum immunoglobulin M (IgM) levels in the WT and apoE−/− mice
Age-dependent increases in the IgM levels were detected in both the WT and apoE−/− mice, significantly higher levels of serum IgM were observed in the apoE−/− mice than in the WT mice throughout the age range (Fig. 2A)
Summary
Based on our previous finding that the apoE deficiency leads to a significant accumulation of pyrP in mice [15], we first examined changes in the serum IgM levels in the WT and apoE−/− mice. To gain insight into the pyrrolation-specific BCR, we sequenced the VH genes of the Pyr+ B-1a cells (pyrBSA-binding CD5+, B220low cells in fraction 4 of Fig. 4C) isolated from PerC of male WT and apoE−/− mice and compared the usage of gene segments selected from the germline gene sets, namely the immunoglobulin heavy chain variable (IGHV), immunoglobulin heavy chain diverse (IGHD), and immunoglobulin heavy chain joining (IGHJ) genes. To compare the characteristics of the HCDR3 region of IgM-BCRs expressed by Pyr+B-1a cells between the male WT and apoE−/− mice, we analyzed IGHV2-2*02, IGHV2-5*01, and IGHV2-9*02, which were found to be the predominant genes in both the WT and apoE−/− mice. SPL cells from male apoE−/− mice at 21 weeks of age were fused with P3/U1 murine myeloma cells, and after screening based on specific binding to pyrBSA, we established three hybridoma clones, 3C8, 5G1, and 5H6, producing IgM
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