PGM3 insufficiency: a glycosylation disorder causing a notable T cell defect

Abstract

Background: Hypomorphic mutations in the phosphoacetylglucosamine mutase 3 (PGM3) gene cause a glycosylation disorder that leads to immunodeficiency. It is often associated with recurrent infections and atopy. The exact etiology of this condition remains unclear.

Objective: This study aimed to characterize the phenotypes and immunological features associated with PGM3 insufficiency and investigate potential disease mechanisms.

Methods: A systematic review of 44 published cases of PGM3 variants was performed, followed by T-cell phenotyping of two patients with PGM3 variants. A genotype-phenotypic severity study was conducted by comparing the residual PGM3 expression of the 12 reconstituted variants in human B cells. A PGM3 inhibitor was used to assess its effect on CD4+ T cell proliferation and differentiation.

Results: Patients with PGM3 variants frequently presented with recurrent infections and atopy, accompanied by reduced naïve CD4+ T cell counts. A genotype-phenotype study showed that low levels of residual PGM3 expression are correlated with disease severity. Notably, inhibition of PGM3 activity impaired TCR-mediated CD4+ T cell proliferation and the synthesis of UDP-GlcNAc, complex N-glycans, O-GlcNAc, glycolytic stress, and mitochondrial respiration during proliferation in a dose-dependent manner. Partial loss of PGM3 activity was observed to preferentially enhance Th1 and Th2 differentiation, while attenuating Th17 and Treg differentiation, consistent with clinical observations.

Conclusion: PGM3 is a critical regulator of CD4+ T-cell proliferation and differentiation. These findings provide new insights into the diverse clinical manifestations and therapeutic development of PGM3 deficiency.

Keywords: CD4+ T cells; PGM3 insufficiency; UDP-GlcNAc; glycosylation; infections.

Figure 1

Potential mechanisms of PGM3 insufficiency causing T-cell defect. In T cells, PGM3 inhibition (in blue) reduces the synthesis of UDP-GlcNAc, which further affects the synthesis and protein modification of complex N-glycans and O-GlcNAc. Furthermore, PGM3 inhibition diminished metabolic pathways, including glycolysis and mitochondrial respiration, during TCR-induced cell proliferation. PPP, pentose phosphate pathway.

Figure 2

Clinical and immunological characterization of PGM3 insufficiency. (A) Clinical manifestations and (B) age of onset in patients with PGM3 mutations. (C) Overall survival of patients with PGM3 insufficiency. (D) Summary of immunological features of patients with PGM3 mutations. (E) T cell phenotyping in family controls and one patient with PGM3 D502Y. (F) Differentiation of Th1 and Th2 subsets from restimulated effector memory (EM) cells derived from one patient identified with PGM3 L83S and one healthy control. RTI, respiratory tract infections; DD, developmental delay; FTT, failure to thrive; GI, gastrointestinal tract infections. EM, effector memory cells; CM, central memory cells; EMRA, effector memory cells re-expressing CD45RA.

Figure 3

Genetic findings of PGM3 insufficiency and the corresponding expression of residual PGM3 protein. (A) Reported mutations and predicted amino acid changes are indicated by the broken lines. Two mutations linked by black lines indicate compound heterozygosity. The numbers in brackets are the number of affected individuals and associated diseases. *E529Q, L480Sfs*10, and D325E reported in the PGM3 isoform 570aa correspond to E501Q, L452Sfs*10, D297E in the PGM3 isoform with 542aa. # Homozygous I322T has been reported in four CID patients, whereas compound heterozygous p. [I322T; R492X] was reported in one SCID patient. (B) Expression of residual PGM3 in BJAB cells reconstituted with WT or mutated PGM3. PGM3-knockout cells were used as the control. The intensity of Western blot bands was quantified by Image J. The ratio of PGM3/Tubulin was calculated and normalized to that of WT PGM3. Data represent more than three independent experiments.

Figure 4

PGM3 inhibition in human T cells suppressed the synthesis of UDP-GlcNAc, protein glycosylation, and metabolic pathways in a dose-dependent manner. Inhibition of PGM3 activity in human primary T cells led to (A) reduced concentration of UDP-GlcNAc, (B) impaired cell proliferation, (C) reduced membrane complex N-glycans (reflected by the intensity of PHA-L staining), (D) reduced protein O-GlcNAcylation, (E) reduced glycolytic rate (left), and mitochondrial respiration (right) in stimulated CD4+ T cells. Pi, PGM3 inhibitor. Each experiment was performed in triplicate or tetraplicate. A repeat experiment was conducted to confirm these results. *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. ns, no significance.

Figure 5

PGM3 inhibition in human naïve CD4+T cells affected T helper (Th) cell differentiation. Inhibition of PGM3 activity in human naïve CD4+T cells caused: (A) enhanced differentiation of Th1 (IFNγ+) and Th2 (IL4+) cells, (B, C) statistical analysis of Th1 and Th2 proliferation, (D, E) reduced Th17 (IL17+) proliferation, and (F, G) reduced Treg differentiation (CD25+Foxp3+) compared to controls. (A, D, F) indicate one of the three experiments corresponding to (B, C, E, G), respectively. Each experiment was performed with triplicates or tetraplicates. A repeat experiment was conducted to confirm these results. *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. ns, no significance.