Abstract
Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species.
Highlights
Protein glycosylation regulates a wide range of processes critical to development and functioning of the central nervous system, including cell adhesion, cellular migration and differentiation, as well as synaptogenesis, synaptic efficacy and plasticity
The analysis of endoglycosidase H (Endo H) or PNGase F treated samples revealed that GluA2 and GluA4 were the only two AMPA receptor subunits sensitive to Endo H or PNGase Fmediated deglycosylation (Figure 1, as we have previously reported in human brain [21])
As we have previously shown in the human [13], we found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F, consistent with large molecular masses of glycans attached to these subunits
Summary
Protein glycosylation regulates a wide range of processes critical to development and functioning of the central nervous system, including cell adhesion, cellular migration and differentiation, as well as synaptogenesis, synaptic efficacy and plasticity. The majority of brain glycoproteins are predicted to be N-glycosylated [2]. Proteins of the mammalian brain have the highest number of predicted N-glycosylation sites and the highest number of tissue specific N-glycosylated proteins [3], suggesting that brain-specific N-glycoproteins coevolved synergistically with its increasing anatomical and functional complexity. The ability to adapt and respond to environmental cues coevolved with the complexity of the mammalian brain, and depends on balanced excitatory synaptic transmission. Glutamate is the major neurotransmitter involved in fast excitatory transmission, which is primarily mediated by the AMPA subtype of glutamate receptor [4,5]. Nglycosylation may play an important role in the trafficking and stabilization of AMPA receptors at the synapse. Glycosylation may affect neurodevelopment: GluA2 in mouse hippocampus expresses the human natural killer-1 (HNK-1) glycol-epitope, which may be essential for dendritic spine morphogenesis in developing neurons [14,15]
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