Altered prion protein glycosylation in the aging mouse brain

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Altered prion protein glycosylation in the aging mouse brain
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  Altered prion protein glycosylation in the aging mouse brain Angeline Xi-Hua Goh,* ,   Chaoyang Li,   Man-Sun Sy,  , § ,1 and Boon-Seng Wong,* ,  , ¶ *  National University Medical Institutes, and Departments of      Biochemistry and   ¶  Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore   Institute of Pathology and   §  Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio,USA Abstract The normal cellular prion protein (PrP C ) is a glycoprotein withtwo highly conserved potential N-linked glycosylation sites. Allprion diseases, whether inherited, infectious or sporadic, arebelieved to share the same pathogenic mechanism that isbased on the conversion of the normal cellular prion protein(PrP C ) to the pathogenic scrapie prion protein (PrP Sc ). How-ever, the clinical and histopathological presentations of priondiseases are heterogeneous, depending not only on thestrains of PrP Sc but also on the mechanism of diseases, suchas age-related sporadic vs. infectious prion diseases. Accu-mulated evidence suggests that N-linked glycans on PrP C areimportant in disease phenotype. A better understanding of thenature of the N-linked glycans on PrP C during the normalaging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. Byusing a panel of 19 lectins in an antibody–lectin enzyme-linkedimmunosorbent assay (ELISA), we found that the lectinbinding profiles of PrP C alter significantly during aging. Thereis an increasing prevalence of complex oligosaccharides onthe aging PrP C , which are features of PrP Sc . Taken together,this study suggests a link between the glycosylation patternson PrP C during aging and PrP Sc . Keywords:  aging, enzyme-linked immunosorbent assay(ELISA), glycosylation, lectin, prion. J. Neurochem.  (2007)  100 , 841–854. The main pathogenic event of prion disease is believed to be the conformational conversion of the  a -helical cellular  prion protein (PrP C ) into the  b -sheet rich scrapie isoformtermed PrP Sc (Prusiner 1998). PrP C is a glycosyl-phospha-tidylinositol (GPI) anchored protein widely expressed inthe brain (Stahl  et al  . 1987), and the protein has twohighly conserved potential sites of   N  -glycosylation (Stim-son  et al  . 1999; Rudd  et al  . 2001; Lawson  et al.  2005).The roles that N-linked glycans play in the functions of PrP C are not completely understood. In cell models, theloss of the glycosylation sites favors the formation of PrPspecies with PrP Sc -like properties (Taraboulos  et al  . 1990;Lehmann and Harris 1997; Neuendorf   et al  . 2004). Theinhibition of glycosylation also enhances the production of PrP Sc in infected cell lines (Taraboulos  et al  . 1990).In one-dimensional (1-D) immunoblots, human- andmouse-brain PrP C are separated into three major bandsthought to be the   35-kDa di-glycosylated, the   32-kDamono-glycosylated and the   28-kDa un-glycosylated iso-forms (Collinge 2001; Pan  et al  . 2002, 2005c). Changes inthe expression profile of these three major glycoformshave been used extensively to distinguish between prionstrains in human (Parchi  et al  . 1997; Collinge 2001) andanimal (Somerville  et al  . 2005) prion diseases. Morerecently, by using monoclonal antibodies (MAbs) that arespecific for distinctive epitopes along the entire PrP C , wefound that the higher molecular weight (MW) band is thefull-length glycosylated protein. In contrast, the two lower  Received May 24, 2006; revised manuscript received July 28, 2006;accepted September 27, 2006.Address correspondence and reprint requests to Dr Boon-Seng Wong, National University Medical Institutes, National University of Singa- pore, MD11 #02–01, 10 Medical Drive, Singapore 117597. Singapore.E-mail: bswong@nus.edu.sg 1 If you would like to request antibodies, please e-mail Dr Man-SunSy: mxs92@case.edu  Abbreviations used  : aa, amino acid; AAL, Aleuria aurantia lectin;CJD, Creutzfeldt–Jakob disease; ConA, Concanavalin A; ELISA,enzyme-linked immunosorbent assay; GalNAc,  N  -acetylgalactosamine;GlcNAc,  N  -acetylglucosamine; GnTIII, GlcNAc transferase-III; GPI,glycosyl-phosphatidylinositol; HRP, horseradish peroxidase; MAbs,monoclonal antibodies; MW, molecular weight; PBS, phosphate-buf-fered saline; PBST, PBS + 0.1% Tween 20; PK, proteinase K; PNGaseF, peptide  N  -glycosidase F; PrP C , cellular prion protein; PrP Sc , scrapie prion protein; rec-moPrP, recombinant mouse PrP.  Journal of Neurochemistry , 2007,  100 , 841–854 doi:10.1111/j.1471-4159.2006.04268.x   2006 The AuthorsJournal Compilation    2006 International Society for Neurochemistry,  J. Neurochem.  (2007)  100 , 841–854  841  MW bands are mostly N-terminally truncated PrP species(Pan  et al  . 2002). Furthermore, by two-dimensional (2-D)immunoblotting, more than 50 PrP species were detected.These species were generated by differential glycosylation,as well as at the sites of proteolytic cleavages. Recently,we found that a common feature of prion diseases inrodents and humans is the accumulation of aberrant full-length PrP species, which bear immature N-linked glycans(Pan  et al  . 2005a,b,c).Comparative analysis of the glycans on hamster PrP C and PrP Sc revealed that PrP Sc has a higher proportion of tri- and tetra-antennary structures than PrP C (Rudd  et al  .1999). Increased quantities of Lewis X and sialyl Lewis X epitopes at both glycosylation sites were also found onmouse PrP Sc (Stimson  et al  . 1999). Enzymatic and lectinanalysis suggest hamster PrP Sc contained complex oligo-saccharides with terminal sialic acids, penultimate galac-toses and fucose residues attached to the innermost   N  -acetylglucosamine (GlcNAc) (Haraguchi  et al  . 1989).At least 52 different glycans structures have been identifiedon PrP C and PrP Sc , but the relative proportion of thesesugars vary between the two isoforms (Rudd  et al  . 1999,2001).Lectins are glycoproteins, each with distinct sugar bindingspecificities similar to the binding of an antibody to anantigen. By using a lectin enzyme-linked immunosorbent assay (ELISA), we found that human PrP C has strongimmunoreactivity with Aleuria aurantia lectin (AAL) andConcanavalin A (ConA) (Pan  et al  . 2002). AAL recognizes a -1,3- and  a -1,6-fucose, and ConA binds to  a -mannoside,indicating that these sugar components are well exposed onhuman PrP C . On the other hand, PrP species in the brains of  patients with Creutzfeldt–Jakob disease (CJD) reacted muchstronger to  Ricinus communis  agglutinin I (RCA I), which preferentially recognizes oligosaccharides ending in galac-tose (Pan  et al  . 2005b).A better understanding of the N-linked glycans on PrP C during normal aging will provide new insights into the pathogenesis of prion diseases. In this study, we used acombination of lectin and enzymatic analysis to examine theglycan pattern on PrP C in the aging mouse brain. Materials and methods Animals The experimental protocols involving laboratory mice were carriedout in accordance with guidelines approved by the InstitutionalAnimal Care and Use Committees (IACUC) at the NationalUniversity of Singapore and Case Western Reserve University.C57BL/6 J mice were killed and brains were collected at postnatalweeks 2, 4, 8, 16, 32 and 64. Three brains ( n  ¼  3 ) from eachtimepoint were used in all analyses. Brains from prion knockout (  Prnp  –/–  ) mice used in this study were obtained as originallydescribed by Bueler   et al  . (1992). Preparation of brain homogenates Brain homogenates (20% w/v) were prepared with lysis buffer, phosphate-buffered saline (PBS; 0.5% Nonidet P-40, 0.5% sodiumdeoxycholate), supplemented with a protease inhibitor cocktail(Roche Diagnostic, Indianapolis, IN, USA) as described (Wong et al  . 2001). The crude brain homogenates were stored at  ) 80  C and protein concentrations were determined by spectrophotometry. Antibodies The generation and characterization of anti-PrP MAbs have been previously described (Zanusso  et al  . 1998; Li  et al  . 2000). TheMAb 8B4, MAb 8H4 and MAb 8F9 used in this study are of theimmunoglobulin G1 subclass, and the epitopes recognized by theseMAbs are diagrammatically presented in Fig. 1. Biotinylation of anti-PrP MAbs was performed using the EZ-linked   Sulfo- N-hydroxysulfosuccinimide-Biotin kit (Pierce, Rockford, IL, USA).Polyclonal antibody to  b -actin was purchased from Sigma (St Louis,MO, USA). Protease digestion and immunoblotting Proteinase K (PK) digestion was performed by incubating total brain homogenate with different concentrations of PK (Sigma) for 1 h at 37  C. The proteolytic reaction was terminated by boiling themixture at 95  C for 10 mins in gel-loading buffer to denature the proteins and was followed by electrophoretic separation on Tris-glycine gels. Immunoblotting was performed following the proce-dure used in an earlier study (Wong  et al  . 2001). The separated proteins were transferred onto a nitrocellulose membrane, probedwith respective antibodies and exposed to horseradish peroxidase(HRP)-conjugated secondary antibodies. The reactive protein bandswere visualized by chemiluminescence using the SuperSignal  West Dura Extended Duration Substrate (Pierce) system. Pre-stained Fig. 1  Anti-prion protein (PrP) monoclonal antibody (MAb) epitopes.The epitopes recognized by the three anti-PrP Mabs, 8B4, 8H4 and8F9, are schematically depicted along the full-length mature mousePrP (MoPrP) sequence (residues 22–232). The MoPrP contains theconserved octarepeats, three  a -helices and two  b -sheets. The two N-linked glycosylation (CHO) sites are located on residue 180 and 196as indicated. 842  A. X.-H. Goh  et al. Journal Compilation    2006 International Society for Neurochemistry,  J. Neurochem.  (2007)  100 , 841–854   2006 The Authors   protein standards (Bio-Rad Laboratories, Hercules, CA, USA) wereused to calculate the apparent molecular weight of the protein bands.The optical densities of the reactive PrP bands, relative to theendogenous  b -actin level, were quantified using the Scion I MAGE (Beta Version 4.0.2, Frederick, MD, USA) software as described(Wong  et al  . 2001). Enzymatic treatment with peptide  N   -glycosidase F andneuraminidase For peptide  N  -glycosidase F (PNGase F) treatment (Pan  et al  .2002), total brain homogenates were mixed with 0.1 volume of 10x denaturing buffer (0.5% SDS, 1%  b -mercaptoethanol) andheated for 10 min at 95  C. PNGase F digestion, as recommended by the supplier (New England BioLabs, Ipswich, MA, USA), wascarried out at 37  C for 1.5 h in 5 m M  sodium phosphate and 1% Nonidet P-40. The endoglycolytic cleavage was terminated by theheat-inactivation of the enzyme at 95  C for 10 min.For neuraminidase (New England Biolabs) digestion (Pan  et al  .2002), total brain homogenates were incubated overnight at 37  C in50 m M  sodium citrate. The exoglycolytic cleavage was terminated by heat-inactivation of the enzyme at 95  C for 10 min.For combined PNGase F and neuraminidase digestion, total brainhomogenates were mixed with 0.1 volume of 10x denaturing buffer (0.5% SDS, 1%  b -mercaptoethanol) and heated for 10 min at 95  C.The mixture was incubated with PNGase F and neuraminidase at 37  C for 4 h in 5 m M  sodium phosphate and 1% Nonidet P-40. Theenzymatic cleavage was terminated by the heat inactivation of theenzyme at 95  C for 10 min. Quantifying brain-derived full-length PrP The quantity of PrP in total brain homogenates was analyzedalongside known quantities of recombinant mouse PrP (rec-moPrP)using sandwich ELISA as previously described (Wong  et al  . 2003).The capturing antibody was MAb 8B4 and 8F9 for full-length andtruncated PrP, respectively, whereas the detecting antibody was biotinylated MAb 8H4. The cloning, expression and purification of rec-moPrP have been described elsewehere (Wong  et al  . 2000). Thecontent of PrP species immuno-captured by either MAb 8B4 or 8F9was calculated from the standard graph of known quantities of recombinant mouse PrP 23–231. Immunoprecipitation Immunoprecipitation of full-length PrP from total brain homo-genates was carried out by equilibrating a predetermined quantity of  brain homogenate with MAb 8B4 at 4  C overnight, as described byPan  et al  . (2002). Protein G agarose beads (Roche Diagnostics)were then allowed to couple to the MAb for 3 h at 4  C. Full-lengthPrP from total brain homogenates were removed by pelleting the beads via centrifugation, leaving truncated PrP in the brainhomogenates remaining in the supernatant fraction. The efficiencyof the immunodepletion process was examined by immunoblotting. Lectin-ELISA An ELISA plate (96-well; Nunc, Naperville, IL, USA) was coatedwith 100 ng of affinity purified either MAb 8B4 or 8F9 overnight at 4  C (Pan  et al  . 2005b) before washing three times with PBST(PBS + 0.1% Tween 20). Unbound sites were blocked with 3% bovine serum albumin (BSA) in PBS and subsequently washed threetimes with PBST. Total brain homogenates were added to theMab 8B4-coated wells, and MAb 8B4-immunodepleted brain hom-ogenates containing truncated PrP species were added to theMAb 8F9-coated wells before incubating them overnight at 4  C.TheplatewaswashedthreetimeswithPBSTbeforeincubationwithavariety of biotinylated lectins (Vector Laboratories, Burlingame, CA, Table 1  Sugar specificity of lectins usedLectins Abbreviation Major Specificity Aleuria Aurantia   Lectin AAL ( a -1,3) or ( a -1,6) fucoseConcanavalin A Con A mannose / glucose Datura Stramonium   Lectin DSL ( b -1,4) linked GlcNAc Erythrina Cristagalli   Lectin ECL galactose / galactosyl ( b -1,4) GlcNAc Galanthus Nivalis   Lectin GNL ( a -1,3) mannose Griffonia Simplicifolia   Lectin I GSL I  a -linked GalNAc / galactose Griffonia (Bandeiraea) Simplicifolia   Lectin II GSL II GlcNAcJacalin Jacalin galactosyl ( b -1,3) GalNAc Lens Culinaris   Agglutinin LCA mannose/glucose Lycopersicon Esculentum   (Tomato) Lectin LEL GlcNAcPeanut Agglutinin PNA galactosyl ( b -1,3) GalNAc Phaseolus vulgaris   Agglutinin PHA-E+L Complex structures (bi-, tri- &tetra-antennary oligosaccharides) Sambucus Nigra   Lectin SNA sialic acid linked ( a -2,6) or ( a -2,3)to terminal galactose Solanum Tuberosum   (Potato) Lectin STL GlcNAc Sophora Japonica   Agglutinin SJA terminal GalNAc / terminal galactoseSuccinylated Wheat Germ Agglutinin SWGA GlcNAc Ulex Europaeus   Agglutinin I UEA I fucose Vicia Villosa   Lectin VVA  a - and  b -linked GalNAc / galactoseWheat Germ Agglutinin WGA GlcNAcGalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine. Aging on prion protein glycosylation  843   2006 The AuthorsJournal Compilation    2006 International Society for Neurochemistry,  J. Neurochem.  (2007)  100 , 841–854  USA) for 1 h at room temperature (25  C). The carbohydratespecificity of our panel of lectins is given in Table 1. Bound lectinswere detected with HRP-conjugated streptavidin (BD Pharmingen,San Jose, CA, USA) incubated at room temperature for 1 h after washingtheplatethreetimesinPBST.Unboundsecondaryantibodieswere removed by washing three times with PBST. Immunoreactivitywas determined by examining the optical density values at 405 nm.The absorbance was measured with 2,2-azino-di-(3-ethylbenzthiaz-oline sulfonic acid) ammonium salt solution (ABTS; Roche Diag-nostics). The results presented are the average of duplicates for individual samples. Statistical analysis Significant differences for the lectin immunoassay were analyzedusing the one-way  ANOVA  followed by Tukey’s Honestly Significant Difference)  post-hoc  test. A  p -value of 0.05 was consideredsignificant. Results Expression profile of PrP species in the aging mouse brain PrP expression was reported to be increased in thedeveloping hamster brain (Sales  et al  . 2002). To determineif PrP expression changes in the aging mouse brain, we prepared total brain homogenates from normal C57BL/6 Jmice at the age of 2, 4, 8, 16, 32 and 64 weeks and thenimmunoblotted them with either MAb 8B4 (Fig. 2a) or MAb 8H4 (Fig. 2b).MAb 8B4, which recognizes an epitope between aminoacid (aa) 37–45 on the mouse PrP sequence (Fig. 1), bindsonly to full-length PrP (Pan  et al  . 2002; Wong  et al  . 2003).At week 2, MAb 8B4 reacts strongly with a major   37-kDa band (band 1) and weakly with a lower MW band (  30 kDa; band 2; Fig. 2a). A third band (  28 kDa; band 3) is onlyapparent after week 8. As MAb 8B4 recognizes only full-length PrP, bands 1, 2 and 3 are likely to be the di-, mono-and un-glycosylated PrP isoforms, respectively (Pan  et al  .2002). Densitometric analysis (Fig. 2c) shows that band 1 isthe predominant band during aging, and that the intensity of  bands 2 and 3 are approximately 15% that of the higher MW band 1.MAb 8H4 recognizes aa 175–185 on mouse PrP (Fig. 1)and can react with both full-length and truncated PrP species(Pan  et al  . 2002; Wong  et al  . 2003). Although the epitoperecognized by MAb 8H4 includes one potential N-linked Fig. 2  Neuronal prion protein (PrP) expression during aging. PrP in40  l g of total brain proteins from 2-, 4-, 8-, 16-, 32- and 64-week-oldwild-type ( n   ¼  3)   mice was immunoblotted with anti-PrP (a) 8B4 and(b) 8H4. The immunoblot is a representation of one animal brainsample from each age group.  b -Actin (bottom) was immunoblotted toensure similar gel loading of the starting material in each sample. Theapparent molecular weight is given in kDa. The optical densities of PrPbands 1 (white), 2 (gray), 3 (black) and 4 (shaded) when immuno-detected by anti-PrP (c) 8B4 and (d) 8H4 in the consecutive-agedbrain samples of wild-type mice were quantified relative to theendogenous  b -actin level for each sample lane in (a) and (b) within aprefixed area using Scion I MAGE  (Beta version 4.0.2) software aspreviously described (Wong  et al  . 2001). Results shown are themean ± SEM from a total of three individual samples per age group. 844  A. X.-H. Goh  et al. Journal Compilation    2006 International Society for Neurochemistry,  J. Neurochem.  (2007)  100 , 841–854   2006 The Authors  glycosylation site, the antibody reacts equally well withrecombinant PrP and native PrP C (Li  et al  . 2000; Pan  et al  .2005b). Hence, this suggests that the presence of N-linkedglycans on native PrP C does not impede the binding of MAb 8H4. Unlike MAb 8B4, MAb 8H4 reacts strongly withthree protein bands (band 1–3; Fig. 2b). Band 1 is likely to be the di-glycosylated full-length PrP isoform. However, asMAb 8H4 binds both full-length and truncated PrP species, band 2 is likely to be a combination of full-length mono-glycosylated and N-terminally truncated PrP isoforms,whereas band 3 may consist of full-length un-glycosylatedand N-terminally truncated PrP isoforms. This heterogeneityof PrP isoforms in bands 2 and 3 may account for the moreintense reactivity (Fig. 2b) observed, as compared withsimilar bands in Fig. 2(a). In Fig. 2(b), we also detected a  15-kDa fragment at week 4 that remains apparent duringaging. This species can only react with MAb 8H4, but not MAb 8B4, and is likely to be the reported N-terminallytruncated C1 fragment found in non-diseased human (Chen et al  . 1995; Jimenez-Huete  et al  . 1998) and mouse (Pan et al  . 2005c) brains. This is because the truncation occursafter the epitope sequence of MAb 8B4 (Chen  et al  . 1995).We have included an actin immunoblot to ensure that thedifferential PrP expression is not the result of unequal proteinloading. Protease sensitivity of PrP species We next examined if the PrP species shows altered proteasesensitivity during aging. Equal quantities of total brain protein from individual mouse brains at different ages weretreated in either the absence or the presence of 0.2, 5, 25 and50  l g/mL PK before immunobloting with MAb 8H4(Fig. 3). At 0.2  l g/mL of PK the level of PrP species at various ages, as defined by the intensity of immunoreactivity,remains essentially unchanged (Fig. 3a). The difference inactin susceptibility to PK digestion is likely to be caused byinconsistent proteolytic efficiency caused by the low enzymeconcentration.After incubation with 5  l g/mL of PK, most PrP speciesare digested. We can only detect residual bands at around  28 kDa remaining in brain samples from weeks 4, 8 and 16(Fig. 3b). Actin is completely digested in all brain samples.The complete digestion of PrP species is only attained after incubating with 25 (Fig. 3c) and 50  l g/mL (Fig. 3d) of PK.In general, the PrP species did not develop proteaseresistance at the PK concentrations used to examine PrP Sc during aging (Pan  et al  . 2005c). Glyco-enzymatic treatment of PrP species In this set of experiments, we incubated the PrP species in brain samples with PNGase F, neuraminidase or a combina- Fig. 3  Neuronal prion protein (PrP) sensitivity to proteinase K (PK)during aging. Brain homogenate PrP in 2-, 4-, 8-, 16-, 32- and 64-week-old wild-type mice was treated either without (–) or with (+) (a)0.2, (b) 5, (c) 25 and (d) 50  l g/mL of PK before immunodetection withanti-PrP 8H4.  b -Actin was immunoblotted as a control. The immuno-blot is a representation of one animal brain sample from each agegroup, and the apparent molecular weight is given in kDa. Aging on prion protein glycosylation  845   2006 The AuthorsJournal Compilation    2006 International Society for Neurochemistry,  J. Neurochem.  (2007)  100 , 841–854
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