The wheat germ agglutinin‐fractionated proteome of subjects with Alzheimer's disease and mild cognitive impairment hippocampus and inferior parietal lobule: Implications for disease pathogenesis and progression

Abstract

Lectin affinity chromatography is a powerful separation technique that fractionates proteins by selectively binding to specific carbohydrate moieties characteristic of protein glycosylation type. Wheat germ agglutinin (WGA) selectively binds terminal N-acetylglucosamine (O-GlcNAc) and sialic acid moieties characteristic of O-linked glycosylation. The current study utilizes WGA affinity chromatography to fractionate proteins from hippocampus and inferior parietal lobule (IPL) from subjects with Alzheimer's disease (AD) and arguably its earliest form, mild cognitive impairment (MCI). Proteins identified by proteomics that were fractionated from MCI and AD hippocampus by WGA affinity chromatography with altered levels compared with age-matched controls included GP96, γ-enolase, glutamate dehydrogenase, glucosidase IIα, 14-3-3ϵ, 14-3-3γ, 14-3-3ζ, tropomyosin-2, calmodulin 2, gelsolin, β-synuclein, α1-antichymotrypsin, and dimethylguanosine tRNA methyltransferase. Proteins identified by proteomics that were fractionated from MCI and AD IPL by WGA affinity chromatography showing altered levels compared with age-matched controls included protein disulfide isomerase, calreticulin, and GP96. The proteins described in this study are involved in diverse processes, including glucose metabolism, endoplasmic reticulum (ER) functions, chaperoning, cytoskeletal assembly, and proteolysis, all of which are affected in AD. This study, the first to use proteomics to identify WGA-fractionated proteins isolated from brains from subjects with MCI and AD, provides additional information about the active proteome of the brain throughout AD progression.

Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice

“…Twenty five μL of total brain homogenate or cell lysate (~100 μg protein) were then added and the plates were incubated for 3 hours at room temperature before being washed three times with 0.2% Tween 20 in PBS. This procedure captures glycosylated proteins containing terminal N-acetylglucosamine modification [31, 32]. Subsequently, an antibody that recognizes O-GlcNAc moieties on the captured proteins (RL2, 1:1000 dilution, Abcam) [33] and S-tagged goat anti-mouse antibody (MSD) were added in 50 μL of 5% Blocker A and 0.2% Tween 20 and the plates were incubated at 4 °C overnight.…”

The role of protein glycosylation in Alzheimer disease

“…WGA and Con A were used to study glycoproteomics in the hippocampus and the inferior parietal lobe in the brains of AD patients, patients with mild cognitive impairment, and nondemented controls [96,97]. Alterations in lectin affinity for proteins involved in, for instance, Glc metabolism (a-enolase, c-enolase, and glutamate dehydrogenase), chaperone functions (Glc-regulated protein 78, heat shock protein 90, protein disulfide isomerase, and Glc-regulated protein 96), cytoskeletal maintenance [GFAP, tropomyosin (TPM)1, TPM2, TPM3, 14-3-3-c,e,f, gelsolin, and calreticulin], synaptic function (dihydropyrimidase 2, Rab GDP dissociation inhibitor XAP4, and b-synuclein) and cell signaling (protein phosphatase-related protein SDS22), and calmodulin) were identified.…”

The 2013 SFRBM discovery award: Selected discoveries from the butterfield laboratory of oxidative stress and its sequela in brain in cognitive disorders exemplified by Alzheimer disease and chemotherapy induced cognitive impairment

“…Other classes of proteins identified by redox proteomics or Western blot methods in our laboratory as oxidatively modified and likely dysfunctional in AD and/or aMCI brain include proteins that normally function as: chaperones [52]; metabolic proteins [53]; proteins involved neurite extension [54]; proteasomal degradation of damaged, aggregated proteins [54]; glutathionylation [55-57] proteins involved in antioxidant defense [27, 28, 45, 47, 58, 59] cell cycle proteins [60, 61] p53-mediated non-transcriptional apoptosis [62, 63] glycoproteins [64]; [65, 66] proteins associated with PSD95 [67]; LRP-1, a major clearance protein that removes Aβ(1-42) from brain to blood [68]; heme oxygenase-1 that degrades pro-oxidant heme forming biliverdin [69]; biliverdin reductase, which rapidly converts biliverdin to the antioxidant and denitrifying molecule, bilirubin, is involved in various pro-survival signaling pathways [70, 71] lipoamide dehydrogenase-mediated reduction of lipoic acid [72]; and protein synthesis [25]. …”