Functionally defective germline variants of sialic acid acetylesterase in autoimmunity

Abstract

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

Figure 1. Analysis of SIAE variants from subjects with autoimmunity

Each SIAE variant found in subjects with autoimmunity was re-created by site-directed mutagenesis in a human SIAE cDNA, that was then sequenced along its entire length. Wild type (WT) SIAE, a known catalytic site mutant (S127A SIAE12), and each SIAE variant that was unique to autoimmune subjects were transfected into 293T cells. Assays were performed for A3G SIAE, N33S SIAE, C196F SIAE, G2121R SIAE, C266G SIAE, Q309P SIAE, T312M SIAE, Y349C SIAE, K400N SIAE, F404S SIAE, and R479C SIAE. Quantitative western blot analysis (using anti-FLAG antibodies) was performed on both the cell lysate and the culture supernatant, and a ratio of these two measurements is shown in the right hand panels of the figure. “Mock” refers to cells which were not transfected but from which lysate and supernatant were analyzed. Half of each lysate was immunoprecipitated with anti-FLAG antibodies and examined for esterase activity, presented following normalization for lysate SIAE protein content. Each row shows results from one representative transfection. Each variant was tested in this manner on at least three or more occasions to ensure reproducibility.

Figure 2. Analysis of SIAE variants from controls

Each variant identified in control subjects was recreated in an SIAE cDNA as described above for subjects with autoimmunity. Wild type (WT) SIAE, S127A SIAE and each SIAE variant that was unique to controls (R62H SIAE, G64S SIAE, Q161K SIAE, R314H SIAE, H447R SIAE, M456I SIAE, and Q462R SIAE) was transfected into 293T cells. Also shown are results from M89V SIAE, which was found in heterozygous form in both patients and controls and in homozygous form only in patients. T312M SIAE was observed in one control and in two patients. Results for this variant are included in Fig. 1. Analyses were performed as described in the legend for Fig. 1.

Figure 3. Analysis of SIAE mutants in terms of secretion, in vitro dominant interfering activity, and effect on induced cell surface 9-O-acetylation of sialic acid

a. Murine C196F Siae, and the murine equivalents of Q309P SIAE and T312M SIAE, (Q335P and T338M Siae), function in a dominant interfering fashion but M89V Siae does not. V5-tagged wild type Siae was transfected along with FLAG-tagged C196F Siae or FLAG-tagged M89V Siae and the enzyme activity of V5-tagged wild type Siae was assessed in transfectants as a function of its protein level. Expression of mutant Siae was monitored by an anti-FLAG Western blot of immunoprecipitated mutant proteins (see Supplementary Fig. 4). b. Pulse-chase analysis comparing secretion of wild type SIAE and M89V SIAE. Transfected 293T cells were metabolically pulse-labeled with ³⁵[S] methionine and lysates and supernatants were immunoprecipitated with anti-FLAG antibodies after 10 minutes, 1 hour, 2 hours and 4 hours of chase. Proteins were separated by SDS-PAGE and revealed by autofluorography. The position of molecular weight markers is indicated on the left in kilodaltons. c. Enhanced 9-O-acetylation of sialic acid following BCR ligation in B cells from a subject with a defective SIAE mutation. Naïve (CD19⁺CD27⁻) B cells from the peripheral blood of a subject with Crohn’s disease (labeled IBD) with a heterozygous SIAE mutation (G212R) and from a control subject were analyzed for cell surface 9-O-acetylation with and without anti-IgM induced BCR ligation. Cell surface 9-O-acetylation was detected using CHE-FcD staining approach as described in Methods. The black tracing reflects CHE-FcD staining and the red represents staining with the second antibody alone.