N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial.

BACKGROUND: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. OBJECTIVES AND METHODS: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). RESULTS: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. CONCLUSIONS: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAc's potential to control residual brain inflammation, myelin repair and neurodegeneration in MS.

Energetic adaptations: Metabolic control of endocytic membrane traffic.

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP-activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell.

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation.

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

Golgi N-glycan branching N-acetylglucosaminyltransferases I, V and VI promote nutrient uptake and metabolism.

Nutrient transporters are critical gate-keepers of extracellular metabolite entry into the cell. As integral membrane proteins, most transporters are N-glycosylated, and the N-glycans are remodeled in the Golgi apparatus. The Golgi branching enzymes N-acetylglucosaminyltransferases I, II, IV, V and avian VI (encoded by Mgat1, Mgat2, Mgat4a/b/c Mgat5 and Mgat6), each catalyze the addition of N-acetylglucosamine (GlcNAc) in N-glycans. Here, we asked whether N-glycan branching promotes nutrient transport and metabolism in immortal human HeLa carcinoma and non-malignant HEK293 embryonic kidney cells. Mgat6 is absent in mammals, but ectopic expression can be expected to add an additional ß1,4-linked branch to N-glycans, and may provide evidence for functional redundancy of the N-glycan branches. Tetracycline (tet)-induced overexpression of Mgat1, Mgat5 and Mgat6 resulted in increased enzyme activity and increased N-glycan branching concordant with the known specificities of these enzymes. Tet-induced Mgat1, Mgat5 and Mgat6 combined with stimulation of hexosamine biosynthesis pathway (HBP) to UDP-GlcNAc, increased cellular metabolite levels, lactate and oxidative metabolism in an additive manner. We then tested the hypothesis that N-glycan branching alone might promote nutrient uptake when glucose (Glc) and glutamine are limiting. In low glutamine and Glc medium, tet-induced Mgat5 alone increased amino acids uptake, intracellular levels of glycolytic and TCA intermediates, as well as HEK293 cell growth. More specifically, tet-induced Mgat5 and HBP elevated the import rate of glutamine, although transport of other metabolites may be regulated in parallel. Our results suggest that N-glycan branching cooperates with HBP to regulate metabolite import in a cell autonomous manner, and can enhance cell growth in low-nutrient environments.

Regulation of human GnT-IV family activity by the lectin domain.

N-Glycan branching critically regulates glycoprotein functions and is involved in various diseases. Among the glycosyltransferases involved in N-glycan branching is the human N-acetylglucosaminyltransferase-IV (GnT-IV) family, which has four members: GnT-IVa, GnT-IVb, GnT-IVc, and GnT-IVd. GnT-IVa and GnT-IVb have glycosyltransferase activity that generates the type-2 diabetes-related ß1,4-GlcNAc branch on the α1,3-Man arm of N-glycans, whereas GnT-IVc and GnT-IVd do not. Recently, this enzyme family was found to have a unique lectin domain in the C-terminal region, which is essential for enzyme activity toward glycoprotein substrates but not toward free N-glycans. Furthermore, interaction between the lectin domain of GnT-IV and N-glycan attached to GnT-IV enables self-regulation of GnT-IV activity, indicating that the lectin domain plays a unique and pivotal role in the regulation of GnT-IV activity. In this review, we summarize the GnT-IV family's biological functions, selectivity for glycoprotein substrates, and regulation of enzymatic activity, with a focus on its unique C-terminal lectin domain.

Regulation of neural stem cell differentiation and brain development by MGAT5-mediated N-glycosylation.

Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating ß1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development.

Age-associated impairment of T cell immunity is linked to sex-dimorphic elevation of N-glycan branching.

Impaired T cell immunity with aging increases mortality from infectious disease. The branching of Asparagine-linked glycans is a critical negative regulator of T cell immunity. Here we show that branching increases with age in females more than males, in naïve more than memory T cells, and in CD4+ more than CD8+ T cells. Female sex hormones and thymic output of naïve T cells (TN) decrease with age, however neither thymectomy nor ovariectomy altered branching. Interleukin-7 (IL-7) signaling was increased in old female more than male mouse TN cells, and triggered increased branching. N-acetylglucosamine, a rate-limiting metabolite for branching, increased with age in humans and synergized with IL-7 to raise branching. Reversing elevated branching rejuvenated T cell function and reduced severity of Salmonella infection in old female mice. These data suggest sex-dimorphic antagonistic pleiotropy, where IL-7 initially benefits immunity through TN maintenance but inhibits TN function by raising branching synergistically with age-dependent increases in N-acetylglucosamine.

The Role of Glycosylation in Melanoma Progression.

Malignant melanoma is the most aggressive form of skin cancer, which originates from the malignant transformation of melanocytes, the melanin-producing cells of the skin. Melanoma progression is typically described as a stepwise process in which metastasis formation ensues late during disease. A large body of evidence has shown that the accumulation of genetic and epigenetic alterations drives melanoma progression through the different steps. Mortality in melanoma is associated with metastatic disease. Accordingly, early-stage melanoma can be cured in the majority of cases by surgical excision, while late-stage melanoma is a highly lethal disease. Glycosylation is a post-translational modification that involves the transfer of glycosyl moieties to specific amino acid residues of proteins to form glycosidic bonds through the activity of glycosyltransferases. Aberrant glycosylation is considered a hallmark of cancer as it occurs in the majority of tumor types, including melanoma. The most widely occurring glycosylation changes in melanoma are represented by sialylation, fucosylation, and N- and I-glycan branching. In this review, we discuss the role of glycosylation in melanoma and provide insights on the mechanisms by which aberrant glycosylation promotes melanoma progression through activation of invasion and metastasis, immune evasion and cell proliferation.

Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy.

Glycoproteins play key roles in various molecular and cellular functions and are among the most difficult to analyze biomolecules on account of their microheterogeneity, non-template-driven synthesis, and low abundances. The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins, including antibodies, vaccines, and biomarkers, are regulated by their glycosylation profile. Thus, there is increasing demand for the qualitative and quantitative characterization and validation of glycosylation on glycoproteins. One of the most important derivatization processes for the structural characterization of released glycans by mass spectrometry (MS) is permethylation. We have recently developed a permethylation strategy in microscale that allows facile permethylation of glycans and permits the processing of large sample sets in nanogram amounts through high-throughput sample handling. Here, we are reporting the wide potential of micropermethylation-based high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells, and purified glycoproteins, through an automated tandem electrospray ionization-mass spectrometry (ESI-MSn) platform. The glycans released from the plasma, cells, and glycoproteins are permethylated in microscale in a 96-well plate or microcentrifuge tube and isolated by a C18 tip-based cleanup through a shorter and simple process. We have developed a workflow to accomplish an in-depth automated structural characterization MS program for permethylated N/O-glycans through an automated high-throughput multistage tandem MS acquisition. We have demonstrated the utility of this workflow using the examples of sialic acid linkages and bisecting GlcNAc (N-acetylglucosamine) on the glycans. This approach can automate the high-throughput screening of glycosylation on large sample sets of glycoproteins, including clinical glycan biomarkers and glycoprotein therapeutics.

Identification of glycan branching patterns using multistage mass spectrometry with spectra tree analysis.

Glycans are crucial to a wide range of biological processes, and their biological activities are closely related to the branching patterns of structures. Different from the simple linear chains of proteins, branching patterns of glycans are more complicated, making their identification extremely challenging. Tandem mass spectrometry (MS2) cannot provide sufficient structural information to deduce glycan branching patterns even with the assistance of various bioinformatic tools and algorithms.The promising technology to identify glycan branching patterns is multi-stage mass spectrometry (MSn). The production-relationship among MSn spectra of a glycan is essentially a tree, making deducing glycan structures from MSn spectra a great challenge. In the present study, we report an approach called glyBranch (glycan Branching pattern identification based on spectra tree) to fully exploit the information contained in the MSn spectra tree for glycan identification. Using 14 glycan standards, including 2 pairs with isomeric sequence, and 16 complex N-glycans isolated from RNase B and IgG, we demonstrated the successful application of glyBranch to branching pattern analysis. The source code of glyBranch is available at https://github.com/bigict/glyBranch/. We have also developed a web-server, which is freely accessible at http://glycan.ict.ac.cn/glyBranch/. SIGNIFICANCE: Glycans are crucial in various biological processes and their functions are closely related to the details of their structures; thus, the identification of glycan branching patterns is of great significance to biological studies. Multistage mass spectrometry (MSn) can provide detailed structural information by generating multiple-level fragments through consecutive fragmentation; however, the interpretation of numerous MSn spectra is extremely challenging. In this study, we present an approach called glyBranch (glycan Branching pattern identification based on spectra tree) to exploit the information contained in MSn spectra tree for glycan identification. This approach will greatly facilitate the automated identification of glycan structures and related biological studies.