Mgat5 deficiency in T cells and experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease initiated by autoreactive T cells. Mgat5, a gene in the Asn (N-) linked protein glycosylation pathway, associates with MS severity and negatively regulates experimental autoimmune encephalomyelitis (EAE) and spontaneous inflammatory demyelination in mice. N-glycan branching by Mgat5 regulates interaction of surface glycoproteins with galectins, forming a molecular lattice that differentially controls the concentration of surface glycoproteins. T-cell receptor signaling, T-cell proliferation, T(H)1 differentiation, and CTLA-4 endocytosis are inhibited by Mgat5 branching. Non-T cells also contribute to MS pathogenesis and express abundant Mgat5 branched N-glycans. Here we explore whether Mgat5 deficiency in myelin-reactive T cells is sufficient to promote demyelinating disease. Adoptive transfer of myelin-reactive Mgat5(-/-) T cells into Mgat5(+/+) versus Mgat5(-/-) recipients revealed more severe EAE in the latter, suggesting that Mgat5 branching deficiency in recipient naive T cells and/or non-T cells contribute to disease pathogenesis.

Figure 1

GlcNAc-branched N-glycan biosynthesis. UDP-GlcNAc is required by the N-acetylglucosaminyltransferases Mgat1, 2, 4, and 5 and iGnT. Cytosolic UDP-GlcNAc enters the Golgi via antiporter exchange with Golgi UMP, a reaction product of the N-acetylglucosaminyltransferases. Galectins bind N-acetyllactosamine, with avidity increasing in proportion to the number of N-acetyllactosamine units (i.e., branching). β-1,6-GlcNAc-branching by Mgat5 promotes poly-N-acetyllactosamine production, further enhancing avidity for galectins. GalT3, galactosyltransferase 3.

Figure 2

Mice deficient in Mgat5 are more susceptible to EAE. (a–e), Splenocytes were isolated from Mgat5^−/− mice 10 days after immunization with MBP + complete Freund's adjuvant (CFA) and restimulated in vitro with MBP for two days. 2.7 million CD3⁺ T cells were injected into naïve Mgat5^+/+ or Mgat5^−/− mice and scored for clinical signs of EAE daily for 40 days. Mice were weighed daily throughout the duration of the experiment and used to determine maximum weight fluctuations. P value for EAE incidence was determined by Fisher's exact test. P values for EAE mean clinical score, disease duration, and the highest clinical score were determined by the Mann-Whitney test.

Figure 3

Mice deficient in Mgat5 have increased IFN-γ and TNF-α levels. Splenocytes were harvested from representative mice from both EAE groups and re-stimulated with 50 ug/mL MPB in vitro. Supernatants from splenocyte cultures were tested for IFN-γ and TNF-α levels by a bead-based analyte detection system using flow cytometry. *P ≤ 0.05. Error bars indicate the means ± S.E. of duplicate samples.