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[Preprint]. 2023 Jun 2:rs.3.rs-2939468.
doi: 10.21203/rs.3.rs-2939468/v1.

IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition

Benjamin Haslund-Gourley  1 , Kyra Woloszcuk  1 , Jintong Hou  1 , Jennifer Connors  1 , Gina Cusimano  1 , Mathew Bell  1 , Bhavani Taramangalam  1 , Slim Fourati  1 , Nathan Mege  1 , Mariana Bernui  1 , Matthew Altman  2 , Florian Krammer  3 , Harm van Bakel  3 , Holden Maecker  4 , Brian Wigdahl  1 , Charles Cairns  5 , Elias Haddad  6 , Mary Comunale  1
Affiliations

IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition

Benjamin Haslund-Gourley et al. Res Sq. .

Update in

  • IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition.
    Haslund-Gourley BS, Woloszczuk K, Hou J, Connors J, Cusimano G, Bell M, Taramangalam B, Fourati S, Mege N, Bernui M, Altman MC, Krammer F, van Bakel H; IMPACC Network; Maecker HT, Rouphael N, Diray-Arce J, Wigdahl B, Kutzler MA, Cairns CB, Haddad EK, Comunale MA. Haslund-Gourley BS, et al. Nat Commun. 2024 Jan 9;15(1):404. doi: 10.1038/s41467-023-44211-0. Nat Commun. 2024. PMID: 38195739 Free PMC article.

Abstract

The glycosylation of IgG plays a critical role during human SARS-CoV-2, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during acute viral infection in humans. In vitro evidence suggests that the glycosylation of IgM inhibits T cell proliferation and alters complement activation rates. The analysis of IgM N-glycosylation from healthy controls and hospitalized COVID-19 patients reveals that mannosylation and sialyation levels associate with COVID-19 severity. Specifically, we find increased di- and tri-sialylated glycans and altered mannose glycans in total serum IgM in severe COVID-19 patients when compared to moderate COVID-19 patients. This is in direct contrast with the decrease of sialic acid found on the serum IgG from the same cohorts. Moreover, the degree of mannosylation and sialylation correlated significantly with markers of disease severity: D-dimer, BUN, creatinine, potassium, and early anti-COVID-19 amounts of IgG, IgA, and IgM. Further, IL-16 and IL-18 cytokines showed similar trends with the amount of mannose and sialic acid present on IgM, implicating these cytokines' potential to impact glycosyltransferase expression during IgM production. When examining PBMC mRNA transcripts, we observe a decrease in the expression of Golgi mannosidases that correlates with the overall reduction in mannose processing we detect in the IgM N-glycosylation profile. Importantly, we found that IgM contains alpha-2,3 linked sialic acids in addition to the previously reported alpha-2,6 linkage. We also report that antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients. Taken together, this work links the immunoglobulin M N-glycosylation with COVID-19 severity and highlights the need to understand the connection between IgM glycosylation and downstream immune function during human disease.

Keywords: COVID-19; Complement Deposition; Glycomics; IgM N-glycan; Immunoglobulin M; SARS-CoV-2.

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Conflict of interest statement

Declarations Competing Interests The authors have no competing interests to declare.

Figures

Figure 1
Figure 1. IgM N-glycosylation analysis reveals differences in COVID-19 patients stratified by trajectory
A) IgM N-glycans labeled with the RapiFluor (RFMS) were profiled with UPLC-FLR-ESI-MS. The resulting N-glycans were identified using mass spectrometry and retention time data. Please see Supplementary Table 1 for a complete list of N-glycans. Dashed lines represent N-glycans without confirmed mass identities due to the limitation of the RFMS label in the QDa mass spectrometer. IgM monomer is displayed with the 5 conserved glycosylation sites labeled B) Cohort demographics: Sex, age, body mass index (BMI), time from symptom onset to hospital admission, and viral load expressed as the delta-delta change between SARS-CoV-2 Nucleocapsid protein 1 (N1) and the house keeping gene RNP via RT qPCR are presented stratified across trajectory 1–2, 3, and 4–5 C) IgM N-glycans are grouped by class: G0 refers to core diantennary N-glycans lacking galactose, G1 refers to core diantennary N-glycans with a single galactose, G2 refers to core diantennary N-glycans with two galactoses, S1 refers to diantennary N-glycans with a single sialic acid, S2 refers to di- and tri-antennary N-glycans with two sialic acids, S3 refers to triantennary N-glycans with three sialic acids, Mannose refers to M4-M10 and hybrid-type N-glycans, Bisecting refers to any N-glycan with a bisecting GlcNAc moiety, Fucosylated refers to any N-glycan with a core-fucose. Healthy Control (n=2), Day 4 Trajectory 1&2 (n=6), Day 7 Trajectory 1&2 (n=5), Day 4 Trajectory 3 (n=6), Day 7 Trajectory 3 (n=5), Day 4 Trajectory 4&5 (n=10), Day 7 Trajectory 4&5 (n=5). N-glycan classes listed in the above graph +/− S.D. with significance denoted were analyzed using a two-way ANOVA with Tukey’s multiple comparisons test **p < 0.01, ***p < 0.001.
Figure 2
Figure 2. IgM N-glycan profile stratifies cohorts of nonsevere from severe trajectory COVID-19 patients
A) IgG N-glycans from healthy control (n=2), day 4 trajectory 1–3 (n=12), and day 4 trajectory 4&5 (n=10) cohorts. N-glycans are graphed as grouped classes – see supplemental figure 4 for a full list of N-glycans and N-glycan grouping. B) IgM N-glycan profiles from cohorts of healthy control (n=2), day 4 trajectory 1–3 (n=12), and day 4 trajectory 4&5 (n=10) hospitalized COVID patients. See Figure 2A for a detailed explanation of the N-glycan classes. C)IgM mannosylated N-glycans from non-severe compared to severe COVID-19. A summation of the indicated mannose/hybrid N-glycan sub-groups are graphed to the right. IgM N-glycan classes graphed as mean +/− S.D. with significance determined using multiple unpaired T-tests *p < 0.05, **p < 0.01, ***p < 0.001
Figure 3
Figure 3. Changes in IgM N-glycosylation correlate with PBMC glycosyltransferase/glycosidase mRNA expression
A) Expression of MAN1A2, MAN2A1, ST3GAL4, and ST6GALNAC2 were significantly different between the COVID-19 trajectory 1–3 (nonsevere) and trajectory 4 and 5 (severe). The role of each glycosidase and glycosyltransferase are depicted below. B) Total mannose on IgM positively correlated with MAN1A2 and TMTC3 expression while negatively correlating with ST3GAL4 expression. The summation of sialic acids on IgM positively correlated with ST3GAL4 expression.
Figure 4
Figure 4. Changes in IgM N-glycosylation Associate with Clinical Markers of COVID-19 Severity
A) Total mannose content (summation of M4-M10 and hybrid N-glycans) was correlated to hospital laboratory measurements of D-dimer, Blood urea nitrogen (BUN), creatinine, and potassium measured on day 4 of hospitalization using linear regression analysis. B) Total di-sialylated (S2) N-glycans were correlated with hospital laboratory measurements of D-dimer, BUN, creatinine, and potassium using simple linear regression. C) Anti-nucleocapsid protein (anti-N) IgA, IgM, and IgG detected from patient plasma donated at the time of hospital admission (Day 0) were correlated to IgM mannose content and S2 content. D) Total IL-18 measured from plasma collected on day 4 of hospitalization was compared between hospitalized trajectories 1–3 and trajectories 4 and 5. IgM mannose and S2 content were correlated with levels of the cytokines IL-16 and IL-18 as detected by Luminex 32-plex assay plasma collected on day 4 of hospitalization. Green dots identify day 4 Trajectory 1+2, yellow dots identify day 4 trajectory 3, and red dots identify day 4 trajectory 4+5 hospitalized COVID-19 cohorts. R2 and p-values are reported below each comparison, with bolded p-values considered statistically significant *p < 0.05 using student’s T-test.
Figure 5
Figure 5. Antigen-specific complement deposition (ADCD) induced by plasma and IgM from severe and nonsevere COVID-19 cohorts
A) Spike S1 and RBD antigen location on SARS-CoV-2 Spike glycoprotein (left), an example of how ADCD assay was quantitated using flow cytometry of plasma compared to PBS-blank sample (center), and the glycosylation of IgM pentamer displaying the c-terminus of IgM containing mannose in orange color while the purple portions of the heavy chain on IgM are complex-type N-glycans (right). B) Gating strategy for detection of complement deposition on fluorescent beads using flow cytometry. C) ADCD assay using the antigens RBD assayed in duplicate with pooled day 4 trajectory 1–3 and day 4 trajectory 4&5 plasma or IgM (left). D) Spike S1 antigen was assayed for ADCD with pooled day 4 trajectory 1–3 and day 4 trajectory 4&5 plasma assayed in triplicate over two experiments and the same cohorts of IgM were assayed in duplicate and then triplicate during a second experiment. E) Total plasma and IgM samples were digested with mannosidase (M) and sialidase (S) before ADCD analysis run in duplicate. Dotted horizontal lines refer to background binding by FITC anti-C3 antibody in PBS-only samples. Statistical significance was analyzed using one-way ANOVA, **p <0.01, ***p <0.001.
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