African-derived genetic polymorphisms in TNFAIP3 mediate risk for autoimmunity

Abstract

The TNF alpha-induced protein 3 (TNFAIP3) is an ubiquitin-modifying enzyme and an essential negative regulator of inflammation. Genome-wide association studies have implicated the TNFAIP3 locus in susceptibility to autoimmune disorders in European cohorts, including rheumatoid arthritis, coronary artery disease, psoriasis, celiac disease, type 1 diabetes, inflammatory bowel disease, and systemic lupus erythematosus (SLE). There are two nonsynonymous coding polymorphisms in the deubiquitinating (DUB) domain of TNFAIP3: F127C, which is in high-linkage disequilibrium with reported SLE-risk variants, and A125V, which has not been previously studied. We conducted a case-control study in African-American SLE patients using these coding variants, along with tagging polymorphisms in TNFAIP3, and identified a novel African-derived risk haplotype that is distinct from previously reported risk variants (odds ratio=1.6, p=0.006). In addition, a rare protective haplotype was defined by A125V (odds ratio=0.31, p=0.027). Although A125V was associated with protection from SLE, surprisingly the same allele was associated with increased risk of inflammatory bowel disease. We tested the functional activity of nonsynonymous coding polymorphisms within TNFAIP3, and found that the A125V coding-change variant alters the DUB activity of the protein. Finally, we used computer modeling to depict how the A125V amino acid change in TNFAIP3 may affect the three-dimensional structure of the DUB domain to a greater extent than F127C. This is the first report of an association between TNFAIP3 polymorphisms and autoimmunity in African-Americans.

FIGURE 1

Haplotype and LD analysis of TNFAIP3 SNPs in African-American SLE cases. A, Using Haploview 4.1 software, the eight haplo-types with frequencies ≥1% in cases and controls were determined. Six of the eight SNPs were genotyped in >90% of our samples and therefore included in the haplotype analysis. Haplotype 2 is the only one that is significantly associated with SLE risk, and only this haplotype contains the minor allele of rs5029953. Haplotype 8 is the protective haplotype, and is the only one carrying the minor allele of rs5029941 (A125V). B, An LD plot generated from African American SLE cases shows the relative linkage between the TNFAIP3 SNPs genotyped in this study. The numbers in each box represent the pairwise r² value × 100 between SNPs, calculated by the Haploview 4.1 software. Darker shaded boxes represent higher r² values between SNPs. The location of each SNP within the general intron/exon structure of the TNFAIP3 locus is indicated above the plot. The 5′ and 3′ untranslated regions are colored dark gray; whereas, the coding regions are colored light gray. The corresponding exon number is indicated above each exonic block.

FIGURE 2

A125V TNFAIP3 mediates diminished degradation of TRAF2. A, The ability of the A125V/F127C double mutant to degrade the protein target TRAF2 was tested. Increasing amounts of either V5-tagged WT or A125V/F127C TNFAIP3 (1, 2.5, or 3.5 μg), or V5-tagged C103A TNFAIP3 (3.5 μg) were cotransfected with HA-tagged TRAF2 (0.5 μg) into HEK293 cells. Whole cell lysates were transferred to PVDF membrane and immunoblotted (IB) for HA, V5, or actin. The A125V/F127C mutant fails to degrade TRAF2 to the same extent as WT TNFAIP3, and is comparable to the known catalytic mutant C103A. Equivalent protein loading was confirmed by immunoblotting for actin. The relative intensity of each protein band was determined, and the densitometry of the TRAF2 and TNFAIP3 bands are plotted using arbitrary units. One of three experiments is shown. B, The ability of TNFAIP3 single mutants to degrade the protein target TRAF2 was tested. Increasing amounts of either V5-tagged WT TNFAIP3 (1, 2.5, 3.5, or 5 μg), V5-tagged C103A TNFAIP3 (1, 2.5, 3.5, or 5 μg), V5-tagged A125V TNFAIP3 (1 or 2.5 μg), V5-tagged F127C TNFAIP3 (1, 2.5, or 3.5 μg), V5-tagged R706Q TNFAIP3 (1, 2.5, 3.5, or 5 μg), or V5-tagged A766P TNFAIP3 (1, 2.5, 3.5, or 5 μg) were cotransfected with HA-tagged TRAF2 (0.5 μg) into HEK 293 cells. Whole cell lysates were transferred to PVDF membrane and IB for HA, V5, or actin. WT TNFAIP3 caused degradation of TRAF2; whereas, the known catalytic mutant C103A TNFAIP3 did not. The A125V, F127C, R706Q, and A766P TNFAIP3 mutants were tested and compared with WT TNFAIP3. Equivalent protein loading was confirmed by immunoblotting for actin. The relative intensity of the protein bands was determined, and the densitometry of the TRAF2 and TNFAIP3 bands are plotted in the lower half of the panel using arbitrary units. On the bar graph for F127C, the highest dose of F127C TNFAIP3 (5 μg) was not determined (*ND). One of three experiments is shown.

FIGURE 3

A125V TNFAIP3 has reduced DUB activity on non–K48-linked polyubiquitinated target proteins. The ability of WT TNFAIP3 and the TNFAIP3 mutants C103A and A125V to deubiquitinate the protein target TRAF2 was tested. Increasing amounts (1, 2.5, 5, or 7.5 μg) of V5-tagged WT TNFAIP3, C103A TNFAIP3, or A125V TNFAIP3 were cotransfected with Myc-tagged K48R-ubiquitin (1 μg) and HA-tagged TRAF2 (3 μg). All three TNFAIP3 constructs have point mutations in conserved cysteines within zinc-fingers 3 and 4 in the C-terminal ubiquitin ligase domain (C607A, C612A, C624A, and C627A), rendering them ubiquitin ligase deficient (E3 ligase deficient). This helped to reduce subsequent degradation of TRAF2 by TNFAIP3. Whole cell lysates were either transferred to PVDF membranes and IB for Myc, HA, V5, or actin (pre-IP), or IP with Ab against HA. These immunoprecipitated lysates were then transferred onto PVDF membranes and immunoblotted for Myc, HA, and V5. The A125V/E3 Ligase mutant demonstrates a reduced ability to DUB TRAF2 in the IP samples, as well as total non–K48-linked polyubiquitinated protein in the pre-IP samples. Equivalent protein loading in the pre-IP samples was confirmed by immunoblotting for actin. The relative intensity of each protein band was determined, and the densitometry of the K48R-Ub and TRAF2 bands in the IP samples are presented on the left side; whereas, the K48R-Ub, TNFAIP3, and TRAF2 bands in the pre-IP samples are plotted on the right side in the lower half of the figure using arbitrary units. Furthermore, a plot showing levels of K48R-Ub in the samples immunoprecipitated for TRAF2 that has been normalized for the amount of TRAF2 present in the IP is located in the lower left bar graph. One of three experiments is shown.

FIGURE 4

The effect of nonsynonymous coding SNPs on the predicted crystal structure in the DUB domain of TNFAIP3. Using MacPyMOL, the reported crystal structure of the DUB domain was used to make the A125V and F127C amino acid changes. PDB accession number 2VFJ was used for this analysis. A, The R group of the valine group at position 125 (pink) has the potential of a steric clash with the R group of the isoleucine at position 29 and/or the isoleucine at position 202 (green). The relative position of the catalytic cysteine at position 103 is indicated in the background in orange. B, The relative positions of the A125V change (purple) pointing into the protein’s core compared with the F127C change (red) pointing away from the protein. The nearby catalytic DUB domain is marked by the cysteine at position 103 (orange).