International Journal of Molecular Sciences Article Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains Frieder Simon 1, Kaya Bork 1, Vinayaga S. Gnanapragassam 1, Tim Baldensperger 2 , Marcus A. Glomb 2 , Simone Di Sanzo 3, Alessandro Ori 3 and Rüdiger Horstkorte 1,* 1 Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg, Hollystr. 1, D-06114 Halle/S., Germany; [email protected] (F.S.); [email protected] (K.B.); [email protected] (V.S.G.) 2 Institut für Chemie—Lebensmittelchemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str.2, D-06120 Halle/S., Germany; [email protected] (T.B.); [email protected] (M.A.G.) 3 Leibniz Institute on Aging—Fritz Lipmann Institut, Beutenbergstr. 11, 07745 Jena, Germany; Simone.DiSanzo@leibniz-fli.de (S.D.S.); Alessandro.Ori@leibniz-fli.de (A.O.) * Correspondence: [email protected] Received: 31 October 2019; Accepted: 2 December 2019; Published: 4 December 2019 Abstract: Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging. Keywords: sialic acid; neural cell adhesion molecule (NCAM), glycation; O-GlcNAc; mannose 1. Introduction Aging is a physiological process resulting in a decline of several functions. The decline of function affects proteins, cells, and organs. One hallmark of molecular aging on protein level is related to posttranslational modifications. Two very prominent posttranslational modifications are glycosylation and glycation—both are related with carbohydrates. Glycosylation represents the attachment of glycans on proteins, which are then called glycoproteins. Proteins are glycosylated during their passage though the ER and the Golgi by a set of enzymes, which build specific glycan structures on serine/threonine (=O-glycan) or asparagine (=N-glycan) residues [1,2]. Glycans on glycoproteins are subdivided into high-mannose- or complex-type structures containing sialic acid (Figure1). With the exception of albumin, most secreted and transmembrane proteins are glycoproteins. In general, glycosylation is important for the folding, the solubility, and the structure of the respective protein, and thereby modulates its function [3]. However, some glycan structures have more specific functions. One example is paucimannose (Figure1), consisting of two N-acetylglucosamine, 1–3 mannose residues, and the Int. J. Mol. Sci. 2019, 20, 6118; doi:10.3390/ijms20246118 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 13 Int. J. Mol. Sci. 2019, 20, 6118 2 of 12 have more specific functions. One example is paucimannose (Figure 1), consisting of two N- acetylglucosamine, 1–3 mannose residues, and the option of a single fucose residue [4]. optionPaucimannose of a single was fucose shown residue to be involved [4]. Paucimannose in the differentiation was shown of to neural be involved progenitor in the cells diff [5].erentiation Another ofrare neural and interesting progenitor glycan cells [ 5of]. the Another brain is rare polysialic and interesting acid (polySia) glycan [6]. of PolySia the brain is nearly is polysialic exclusively acid (polySia)expressed [ 6on]. PolySiathe neural is nearlycell adhesion exclusively molecule expressed NCAM on (neural the neural cell adhesion cell adhesion molecule). molecule Expression NCAM (neuralof polySia cell interferes adhesion with molecule). the adhesion Expression between of polySia cells by interferes introducing with negative the adhesion charges between and increasing cells by introducingthe bound water negative [7,8]. charges PolySia and is increasingessential for the normal bound brain water development, [7,8]. PolySia but is essential also during for normal cancer brainprogression development, of NCAM-expressing but also during tumors cancer [9]. progression However, of there NCAM-expressing are many more tumors functions [9]. However,of glycan therestructures, are many which more cannot functions all be ofsummarized glycan structures, in one single which introduction. cannot all be Disturbed summarized glycosylation in one single by introduction.genetic defects Disturbed in the biosynthesis glycosylation of glycan by geneticstructur defectses are called in the congenital biosynthesis disorders of glycan of glycosylation structures areand called lead to congenital dramatic disordersphenotypes, of glycosylationincluding mental and retardation, lead to dramatic indicating phenotypes, the importance including of mentalcorrect retardation,glycosylation indicating for the thefunction importance of the of brain correct and glycosylation other organs for the[10]. function Attachment of the brainof a andsingle other N- organsacetylglucosamine [10]. Attachment (O-GlcNAc) of a single to serine N-acetylglucosamine or threonine residues (O-GlcNAc) on intracellular to serine orproteins, threonine so called residues O- onGlcNAcylation, intracellular proteins, represent so a calleddynamicO-GlcNAcylation, glycosylation (Figure represent 1) [11]. a dynamic Reversible glycosylation O-GlcNAcylation (Figure1 )[opens11]. Reversiblethe possibilityO-GlcNAcylation to regulate protein opens or the enzyme possibility functi toon regulate similar to protein protein or phosphorylation. enzyme function similarHowever, to proteinlittle is known phosphorylation. on the role However, of glycosylation little is during known aging. on the role of glycosylation during aging. FigureFigure 1.1. StructureStructure ofof thethe analyzedanalyzed glycans.glycans. On the leftleft panel, some typical N-glycans are shown:shown: High-mannoseHigh-mannose structures (From (From Man5 Man5 to to Man9), Man9), wh whichich are are assembled assembled in the in ER, the whereas ER, whereas sialicsialic acid- acid-containingcontaining complex complex structures structures are generated are generated in the inGolgi. the Golgi.The right The panel right shows panel O shows-GlcNAcO-GlcNAc attached attachedon serine on or serinethreonine or threonine residues residuesin the cytosol in the by cytosol O-GlcNAc-transferase by O-GlcNAc-transferase (OGT). (OGT). GlycationGlycation isis aa non-enzymaticnon-enzymatic chemicalchemical reactionreaction betweenbetween thethe carbonylcarbonyl moietymoiety ofof aa reducingreducing sugarsugar oror ofof dicarbonylsdicarbonyls [[12],12], suchsuch asas methylglyoxalmethylglyoxal oror glyoxal,glyoxal, whichwhich areare derivedderived duringduring glycolysisglycolysis oror lipidlipid peroxidation,peroxidation, andand aa freefree aminoamino groupgroup ofof aa protein.protein. FurtherFurther degradationdegradation ofof thesethese productsproducts resultsresults inin thethe formationformation ofof advancedadvanced glycationglycation endproductsendproducts (AGEs)(AGEs) [[13,14].13,14]. FormationFormation of AGEsAGEs cancan leadlead toto proteinprotein aggregationaggregation or or misfunction, misfunction, and and accumulation accumulation of of AGE-modified AGE-modified proteins proteins is associated is associated with with the progressionthe progression of several of diseasesseveral (diabetes,diseases Alzheimer’s (diabetes, disease,Alzheimer’s multiple disease, sclerosis, multiple or atherosclerosis) sclerosis, [ 15or]. AGEsatherosclerosis) can also be [15]. recognized AGEs can by also some be cellularrecognized receptors. by some The cellular best characterizedreceptors. The receptor best characterized for AGEs isreceptor the receptor for AGEs for advancedis the receptor glycation for advanced endproducts glycation (RAGE) endproducts [16]. Binding (RAGE) of AGEs[16]. Binding to RAGE of AGEs leads toto internalizationRAGE leads to and internalization signal transduction and signal [17], transduction resulting in activation[17], resulting of p44 in/p42 activation mitogen-activated of p44/p42 proteinmitogen-activated kinase and nuclearprotein kinase transcription and nuclear factor transcriptionκB (NF-κB) [18 factor]. κB (NF-κB) [18]. TheThe aimaim ofof thisthis studystudy waswas toto demonstratedemonstrate thatthat specificspecific posttranslationalposttranslational modificationsmodifications correlatecorrelate withwith aging.aging. We We chose chose the the brain, brain, since since aging aging is encompassed is encompassed with with a variety a variety of changes, of changes, which arewhich related are torelated declined to declined brain function. brain function. Therefore, Therefore, we prepared we