Neuroscience Letters 367 (2004) 235–240 Glycosylation changes in Alzheimer’s disease as revealed by a proteomic approach Katja Kanninen a , Gundars Goldsteins a , Seppo Auriola b , Irina Alafuzoff c , Jari Koistinaho a,d,∗ a Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Neulaniementie 2, P.O. Box 1627, FIN-70211 Kuopio, Finland b Department of Pharmaceutical Chemistry, University of Kuopio, 70211 Kuopio, Finland c Department of Neuroscience and Neurology, Kuopio University Hospital, University of Kuopio, 70211 Kuopio, Finland d Department of Oncology, Kuopio University Hospital, 70211 Kuopio, Finland Received 16 April 2004; received in revised form 3 June 2004; accepted 4 June 2004 Abstract Glycosylation influences the biological activity of proteins and affects their folding and stability. Because aberrant glycosylation is associated with Alzheimer’s disease (AD), we applied proteome analysis together with Pro-Q Emerald 300 glycoprotein staining to investigate changes in glycosylated cytosolic proteins in AD and control brain. Frontal cortex proteins from 10 AD patients and 7 non-demented controls were subjected to separation by two-dimensional gel electrophoresis and subsequently stained with carbohydrate-specific Pro-Q Emerald 300 dye. Changes in glycosylation of separated proteins were quantified, and proteins of interest identified by mass spectrometry. Approximately 30% of all detectable proteins in the human frontal cortex appeared glycosylated, including heat shock cognate 71 stress protein and beta isoform of creatine kinase. The glycosylation of collapsin response mediator protein 2 (CRMP-2) and an unknown protein was reduced in AD, while the glycosylation of glial fibrillary acidic protein was increased. CRMP-2 regulates the assembly and polymerization of microtubules and is associated with neurofibrillary tangles in AD. Aberrant glycosylations in AD may help understand the mechanisms of neurodegenerative diseases. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Glycosylation; Frontal cortex; Collapsin response mediator protein 2; Glial fibrillary acidic protein Post-translational modifications determine the functions of proteins and play a crucial role not only in normal devel- opment of the nervous system [7] but also in brain injury and neurodegeneration, including Alzheimer’s disease (AD) [3–5,10,17,24]. Probably the most studied post-translational modification is phosphorylation, which evidently is a key mechanism in intracellular signal transduction and in some neuropathologies, such as formation of neurofibrillary tan- gles (NFT) [17], a hallmark of AD. Even though protein gly- cosylation is estimated to be a more abundant modification than protein phosphorylation in various tissues, very little is known about glycosylated proteins in the human brain. Re- cently, altered glycosylation of tau [17], cholinesterases [21] and transferrin [24] in AD have been described, suggesting that aberrant glycosylation changes occur in AD. This view ∗ Corresponding author. Tel.: +358 17 162427; fax: +358 17 163030. E-mail address: jari.koistinaho@uku.fi (J. Koistinaho). is also supported by the observations that the amount of O-glycosylated proteins is increased in neuritic plaques [10] and neurofibrillary tangles of this disease. The functional importance of glycosylation in AD is underlined by the find- ings that glycosylation regulates nicastrin [26] and Asp-2 [5], a presenilin complex component and a beta-secretase protein, respectively, which play a role in cleavage of the amyloid precursor protein. Moreover, aberrant glycosylation of tau may be involved in neurofibrillary degeneration by fa- cilitating abnormal hyperphosphorylation of tau [17]. Here we applied proteome analysis together with Pro-Q Emerald 300 glycoprotein staining to screen whether proteins, which have been reported to show alterations in total protein ex- pression, oxidation or phosphorylation, show changes also in protein glycosylation. The experimental protocol was approved by the Ethi- cal review committee of the Kuopio University Hospital and meets the guidelines of the European Union. Frontal cortex samples from 7 neurologically unimpaired and 10 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.06.013