Autoantibodies against SUMO1-DHX35 in long-COVID

Abstract

Long COVID is a collection of symptoms as a late sequelae of SARS-CoV-2 infection. It often includes mental symptoms such as cognitive symptoms, persisting loss of smell and taste, in addition to exertional dyspnea. A role of various autoantibodies (autoAbs) has been postulated in long-COVID and is being further investigated. With the goal of identifying potentially unknown autoAbs, we screened plasma of patients with long COVID on in-house post-translationally modified protein macroarrays including citrullinated, SUMOylated and acetylated membranes. SUMO1ylated isoform DEAD/H (Asp-Glu-Ala-Asp/His) box helicase 35 (SUMO1-DHX35) was identified as only candidate antigen. In adult patients with long COVID, IgG autoAbs against SUMO1-DHX35 of IgG class were found in seven of 71 (9.8%) plasma samples, of IgM and IgG class in one of 69 (1.4%) samples, not in 200 healthy adult controls, not in 442 healthy children, and 146 children after SARS-CoV-2 infection. All autoAb-positive seven patients were female. AutoAb titers ranged between 200 to up to 400 By point mutagenesis and expression of FLAG-tagged mutants of DHX35 in HEK293 cells, and subsequent SUMOylation of purified constructs, lysine 53 was identified as a unique, never yet identified, SUMOylation site. The autoAbs had no reactivity against the non-SUMO1ylated mutant (K53R) of DHX35. To summarize, autoAbs against SUMO1-DHX35 were identified in adult female patients with long-COVID. Further studies are needed to verify the frequency of occurrence. The function of DHX35 has not yet been determined and there is no available information in relation to disease implication. The molecular mechanism causing the SUMOylation, the potential functional consequences of this post-translational modification on DHX35, and a potential pathogenicity of the autoAbs against SUMO1-DHX35 in COVID-19 and other possible contexts remain to be elucidated.

Fig. 1

A) ELISA of plasma samples of long-COVID-19 against DHX-35 and SUMO1-DHX35. OD values were measured at 490 nm. B) Titers of anti-SUMO1-DHX35 antibodies. What is shown is not titers but OD values. C) Ig classes of Anti-SUMO1-DHX35-antibodies.

Fig. 2

A) Western blots of different point-mutated, full-length and C-terminally FLAG-tagged constructs of DHX-35, which were either treated post-translationally to be SUMO1ylated or not. Using anti-FLAG or anti-DHX35 as primary antibodies demonstrates gain of molecular weight due to added SUMO1-group in all mutants but K53R. SUMO1-specific primary antibody confirms K53 as SUMO1-site. B) ELISA using different point-mutated, full-length and C-terminally FLAG-tagged constructs of DHX-35 as antigens. These where were either used directly (above) or treated post translationally by SUMO1ylation (below). Anti-FLAG, anti-SUMO1, or plasma of long-COVID patients were used. C) Western blot of DHX35 of whole blood cell lysate using either anti-SUMO1- or commercially available anti-DHX35 antibodies. Blood cell lysates were derived of anti-SUMO1-DHX35-antibody positive and negative patients.