Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma), Madrid, Spain.
Technion-Israel Institute of Technology, Haifa, Israel.
Arthritis Rheumatol. 2016 Oct;68(10):2466-75. doi: 10.1002/art.39734.
To determine the influence of endoplasmic reticulum aminopeptidase 2 (ERAP-2) expression on the HLA-B*27 peptidome in live cells.
Using immunoaffinity chromatography and acid extraction, HLA-B27:05-bound peptides were isolated from 2 ERAP-2-negative lymphoblastoid cell lines and 1 ERAP-2-positive lymphoblastoid cell line expressing functionally indistinguishable ERAP-1 variants. More than 2,000-4,000 B27:05 ligands were identified from each cell line, and their relative abundance was established by quantitative tandem mass spectrometry and MaxQuant-based peptide analyses. Pairwise comparisons were used to determine the structural features of peptides whose relative abundance was dependent on the presence of ERAP-2. Synthetic peptide digestions were performed with recombinant ERAP-1 and ERAP-2. Peptide affinity was estimated with standard algorithms.
The B27:05 peptidome from ERAP-2-positive cells showed 3-4% fewer peptides with N-terminal basic residues than did the peptidome from ERAP-2-negative cells. Among the shared peptides, those most abundant in the presence of ERAP-2 included more nonamers, fewer decamers, and fewer N-terminal basic residues than the peptides predominant in ERAP-2-negative cells. These ERAP-2-dependent changes did not alter the global affinity of the B27:05 peptidome.
ERAP-2 significantly influences the B*27:05-bound peptidome by destroying some ligands and decreasing the abundance of many more ligands with N-terminal basic residues, while increasing the abundance of nonamers. The former effects are best explained by direct ERAP-2 trimming. The effects on peptide length might be attributed to ERAP-2-induced activation of ERAP-1 trimming. These data support the notion of a peptide-mediated mechanism as the basis for the association of ERAP-2 with ankylosing spondylitis. Analogous effects on other major histocompatibility complex class I peptidomes might explain the involvement of ERAP-2 in HLA-B27-negative spondyloarthritis.
确定内质网氨肽酶 2（ERAP-2）表达对活细胞中 HLA-B*27 肽组的影响。
使用免疫亲和层析和酸提取，从 2 个 ERAP-2 阴性淋巴母细胞系和 1 个表达功能上无差异的 ERAP-1 变体的 ERAP-2 阳性淋巴母细胞系中分离 HLA-B27:05 结合肽。从每个细胞系中鉴定出 2000 至 4000 多个 B27:05 配体，并通过定量串联质谱和基于 MaxQuant 的肽分析确定其相对丰度。使用成对比较确定相对丰度依赖于 ERAP-2 存在的肽的结构特征。使用重组 ERAP-1 和 ERAP-2 进行合成肽消化。使用标准算法估计肽亲和力。
与 ERAP-2 阴性细胞相比，ERAP-2 阳性细胞的 B27:05 肽组中 N 端碱性残基的肽少 3-4%。在共同的肽中，与 ERAP-2 存在时最丰富的肽包括更多的非九肽，更少的十肽，以及比 ERAP-2 阴性细胞中主要的肽更少的 N 端碱性残基。这些 ERAP-2 依赖性变化并未改变 B27:05 结合肽组的整体亲和力。
ERAP-2 通过破坏一些配体并减少许多具有 N 端碱性残基的配体的丰度，同时增加非九肽的丰度，从而显著影响 B*27:05 结合肽组。直接的 ERAP-2 修剪可最好地解释前一种作用。对肽长度的影响可能归因于 ERAP-2 诱导的 ERAP-1 修剪的激活。这些数据支持肽介导机制作为 ERAP-2 与强直性脊柱炎相关的基础。类似的作用可能解释了 ERAP-2 在 HLA-B27 阴性脊柱关节炎中的参与。
