Journal of Neuroscience Research 88:3566–3577 (2010) 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 Fabio Di Domenico,1 Joshua B. Owen,2 Rukhsana Sultana,2 Rena A. Sowell,2 Marzia Perluigi,1 Chiara Cini,1 Jian Cai,3 William M. Pierce,3 and D. Allan Butterfield2* 1Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy 2Department of Chemistry, Center of Membrane Sciences, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 3Department of Pharmacology, University of Louisville, Louisville, Kentucky Lectin affinity chromatography is a powerful separation Alzheimer’s disease (AD) is a neurodegenerative dis- techniquethatfractionatesproteinsbyselectivelybinding order that currently affects 5.3 million Americans and in to specific carbohydrate moieties characteristic of protein the absence of interventions to slow or halt progression of glycosylation type. Wheat germ agglutinin (WGA) selec- this dementing disorder is predicted to affect 16–20 mil- tively binds terminal N-acetylglucosamine (O-GlcNAc) and lion Americans in just a few decades (Mebane-Sims, sialic acid moieties characteristic of O-linked glycosyla- 2009). AD is the most common cause of dementia and is tion. The current study utilizes WGA affinity chromatogra- characterized pathologically by the occurrence of neurofi- phy to fractionate proteins from hippocampus and inferior brillary tangles (NFTs) and senile plaques (SPs) in the neo- parietal lobule (IPL) from subjects with Alzheimer’s disease cortex, entorhinal cortex, and hippocampus (Markesbery, (AD) and arguably its earliest form, mild cognitive impair- 1997). In addition, the aforementioned brain regions as ment (MCI). Proteins identified by proteomics that were well as hippocampal efferents to the inferior parietal lobule fractionated from MCI and AD hippocampus by WGA af- (IPL) are dramatically atrophied in AD brain compared finity chromatography with altered levels compared with with the brains from nondemented subjects (Clower et al., age-matched controls included GP96, g-enolase, gluta- 2001). NFTs occur intracellularly and feature paired heli- mate dehydrogenase, glucosidase IIa, 14-3-3e, 14-3-3g, cal filament (PHF) structures composed of hyperphos- 14-3-3f, tropomyosin-2, calmodulin 2, gelsolin, b-synu- phorylated tau, whereas senile plaques are extracellular clein, a1-antichymotrypsin, and dimethylguanosine tRNA and are composed primarily of amyloid-beta peptides methyltransferase. Proteins identified by proteomics that (Abs). Abs contained in SPs range between 39 and 43 were fractionated from MCI and AD IPL by WGA affinity amino acids in length, the Ab 1–42 variant being the chromatography showing altered levels compared with dominant form present in the plaques (Selkoe, 2004). age-matched controls included protein disulfide isomer- Amnestic mild cognitive impairment (aMCI) is arguably ase, calreticulin, and GP96. The proteins described in this study are involved in diverse processes, including glucose metabolism, endoplasmic reticulum (ER) functions, chap- Fabio Di Domenico and Joshua B. Owen contributed equally to this work. eroning, cytoskeletal assembly, and proteolysis, all of Contract grant sponsor: NIH; Contract grant number: AG-05119 (to which are affected in AD. This study, the first to use pro- D.A.B.); Contract grant number: AG-10836 (to D.A.B.); Contract grant sponsor: Istituto Pasteur—Fondazione Cenci Bolognetti (to F.D.D.). teomics to identify WGA-fractionated proteins isolated from brains from subjects with MCI and AD, provides addi- *Correspondence to: Prof. D. Allan Butterfield, Department of Chemis- tional information about the active proteome of the brain try, Center of Membrane Sciences, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506. E-mail: [email protected] throughout AD progression. VC 2010 Wiley-Liss, Inc. Received 15 June 2010; Revised 28 August 2010; Accepted 3 September Key words: proteomics; wheat germ agglutinin; lectin 2010 affinity chromatography; mass spectrometry; Alzheimer’s Published online 8 October 2010 in Wiley Online Library disease; amnestic mild cognitive impairment (wileyonlinelibrary.com). DOI: 10.1002/jnr.22528 ' 2010 Wiley-Liss, Inc. WGA-Fractionated Brain Proteome in AD and MCI 3567 the earliest form of AD. aMCI patients have detectable TABLE I. MCI and AD Subject Profiles deficits in speech, memory, or other essential cognitive Brain abilities but no dementia; however, these deficits do not weight interfere with activities of daily living (Mebane-Sims, Age (years) Sex (g) PMI (hr) Braak stage 2009). Estimates suggest that 10–20% of the population aged 65 and older has aMCI, and a significant portion of Control 1 93 Female 1,080 2.75 2 Control 2 74 Male 1,140 4.00 1 aMCI patients progresses further to AD (Mebane-Sims, Control 3 86 Female 1,150 1.75 1 2009). The clinical involvement of patients diagnosed Control 4 76 Female 1,315 2.00 1 with aMCI is a vital focus of AD research. Control 5 79 Male 1,240 1.75 2 Protein glycosylation is a posttranslational modifi- Control 6 86 Female 1,300 3.75 1 cation (PTM) that affects proteins in a variety of Average 82 6 7.2 1,204 6 95 2.67 6 1.01 1.33 6 0.52 ways, including proper protein folding, protein function MCI regulation, and cellular localization. Protein glycosyla- MCI 1 99 Female 930 2.00 5 tion occurs in two general forms, N-linked glycosyla- MCI 2 88 Female 1,080 2.25 5 tion and O-linked glycosylation. N-linked glycosylation MCI 3 87 Male 1,200 3.50 4 occurs in the endomembrane system and has a can- MCI 4 87 Male 1,170 2.25 3 MCI 5 91 Female 1,155 5.00 3 onical serine/threonine (Ser/Thr)-X-asparagine (Asn) MCI 6 82 Female 1,075 3.00 3 motif to which glycans are added to the Asn residue. Average 89 6 5.7 1,102 6 98 3.0 6 1.1 3.8 6 1.0 O-linked glycosylation is the addition of carbohydrates Control 1 77 Male 1,310 3.50 1 to Ser or Thr residues; however, O-linked glycosyla- Control 2 83 Male 1,275 2.00 3 tion sites do not have a well-characterized motif for Control 3 87 Male 1,150 2.00 2 glycan addition. O-linked glycosylation has traditionally Control 4 72 Male 1,150 3.75 1 been thought to occur only in the Golgi apparatus Control 5 85 Female 1,020 2.50 3 as part of the secretory pathway, but studies show that Control 6 81 Male 1,410 2.00 2 O-linked glycosylation occurs on cytoplasmic and nu- Average 81 6 5.5 1,219 6 139 2.60 6 0.8 2.00 6 0.9 cleoplasmic proteins through the addition of a single AD 1 80 Female 1,160 2.75 6 AD 2 90 Female 1,050 2.60 6 N-acetylglucosamine (O-GlcNAc) residue to Ser/Thr AD 3 88 Male 1,230 5.75 5 (Hart, 1997; Hart et al., 2007). The exact role of non- AD 4 81 Male 1,260 3.00 6 secretory pathway O-linked glycosylation is unknown, AD 5 81 Female 835 3.00 6 but studies suggest that nucleoplasmic and cytoplasmic AD 6 92 Female 1,090 2.00 6 O-linked glycosylation works in concert with phospho- Average 85 6 5.3 1,104 6 154 3.2 6 1.3 5.8 6 0.4 rylation to regulate proteins (Hart et al., 2007). Addi- tionally, nonsecretory pathway O-link glycosylation has been hypothesized to function as a cellular glucose sensor (Lefebvre et al., 2010). Levels of nonsecretory MATERIALS AND METHODS pathway O-linked glycosylation are decreased globally Sample Preparation in AD, including levels of O-GlcNAc modified tau 5 protein (Liu et al., 2004). Hippocampus and IPL samples (n 6 each) from well- Lectin affinity chromatography is a powerful separa- characterized subjects with AD and MCI and age-matched con- tion technique that fractionates proteins by selectively trols (Table I) were obtained from the University of Kentucky binding to specific carbohydrate moieties characteristic of Alzheimer’s Disease Clinical Center Neuropathology Core. protein glycosylation type. Concanavalin-A (ConA) and Samples were homogenized on ice with sucrose isolation buffer [0.32 M sucrose with protease inhibitors, 4 lg/ml leupeptin, wheat germ agglutinin (WGA) are the two most widely l l used lectins for chromatographic separations. ConA has af- 4 g/ml pepstatin A, 0.125 M Tris, pH 8.0, 5 g/ml aprotinin, finity for high-mannose and terminal glucose carbohy- 0.2 mM phenylmethylsulfonylfluoride (PMSF), and 0.6 mM drates that are commonly found in N-linked glycopro- MgCl2]. Table I presents demographic data for the MCI and teins. In addition, ConA contains a binding site with an AD subjects. Note the very short post-mortem interval of affinity for excessively hydrophobic proteins. WGA selec- these samples. tively binds terminal N-acetylglucosamine (O-GlcNAc) and sialic acid moieties characteristic of O-linked glycosy- Protein estimation and WGA Lectin Affinity Columns lation. The addition of O-GlcNAc occurs in both secre- Protein concentrations were determined using the BCA tory and nonsecretory O-linked glycosylation. Previous method, and aliquots (1,500 lg) were diluted with 53 binding/ studies from our laboratory utilized ConA to fractionate wash buffer in a 4:1 volumetric ratio and loaded into WGA af- proteins from MCI and AD hippocampus and inferior finity columns. WGA lectin columns, binding/wash, and elu- parietal lobule (IPL; Owen et al., 2009). The current tion buffers were prepared as described in the manufacturer’s study explores the glycoproteome of MCI and AD hippo- instructions (Pierce Biotechnology, Rockford IL). The samples campus and IPL using WGA lectin affinity chromatogra- were loaded onto columns and incubated for 10 min with end- phy coupled to two-dimensional (2D) gel proteomics over-end mixing using an elliptical rotor.