Báo cáo khoa học: Ligand binding promotes prion protein aggregation – role of the octapeptide repeats potx

Ligand binding promotes prion protein aggregation – role of the octapeptide repeats Shuiliang Yu1, Shaoman Yin1, Nancy Pham2, Poki Wong1, Shin-Chung Kang1, Robert B Petersen1, Chaoyang Li1 and Man-Sun Sy1 Department of Pathology, Case Western Reserve University, Cleveland, OH, USA Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA Keywords aggregation; copper; glycosaminoglycan; octapeptide repeat; prion Correspondence M.-S Sy, Room 5131 Wolstein Research Bldg, School of Medicine, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106-7288, USA Fax: +1 216 368 1357 Tel: +1 216 368 1268 E-mail: man-sun.sy@case.edu (Received July 2008, revised 18 August 2008, accepted 10 Sepember 2008) doi:10.1111/j.1742-4658.2008.06680.x Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu2+ and Zn2+ Here, we report our findings that GAG and Cu2+ promote the aggregation of recombinant human PrP (rPrP) The normal cellular prion protein has five octapeptide repeats In the presence of either GAG or Cu2+, mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu2+ is abolished By contrast, when the Cu2+-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu2+ is able to promote aggregation Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu2+-promoted aggregation However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu2+ Therefore, although binding of either GAG or Cu2+ promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation Prion diseases are a group of fatal neurodegenerative disease in humans and animals It is believed that all prion diseases are caused by the conversion of a normal cellular prion protein (PrPC) to a pathogenic and infectious isoform, commonly referred to as scrapie prion (PrPSc) or proteinase-resistant prion (PrPRES) [1] The majority of human prion diseases are sporadic, and the cause of the disease is not known A small number of prion diseases, such as Kuru, iatrogenic Creutzfeldt–Jacob disease and variant Creutzfeldt– Jacob disease are contracted through an infectious mechanism By contrast, familial or inherited human prion disease, which accounts for  10–15% of human prion diseases, is the result of mutations in the germline prion protein gene, PRNP More than 30 different pathogenic mutations in human PRNP have been identified [2,3] These mutations are either insertional or point mutations The insertion mutations occur solely in the octapeptide-repeat region; wild-type human PrP has five octapeptide repeats The number of pathogenic insertions ranges from two to nine However, point mutations occur along the entire PrP molecule, but tend to cluster in the C-terminal globular domain It is thought that the mutant prion protein is inherently Abbreviations GAG, glycosaminoglycan; PBST, NaCl ⁄ Pi ⁄ 0.05% Tween; PrP, prion protein; PrPC, normal cellular form of PrP; PrPSc, the infectious and pathogenic scrapie PrP; rPrP, recombinant wild-type PrP; rPrPD51-90, recombinant PrP with deletion of octapeptide-repeat region; rPrPDKKRPK, recombinant PrP with deletion of GAG binding motif, KKRPK at the beginning of the N-terminal; rPrP10OR, recombinant PrP with 10 octapeptide-repeats; rPrP8OR, recombinant PrP with octapeptide-repeats 5564 FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS S Yu et al unstable, prone to misfold and aggregate, forming a structure which acts as a ‘seed’ to recruit additional mutant proteins, eventually leading to the formation of pathogenic and infectious PrPSc [4] Recombinant bacterial-produced wild-type PrP, rPrP and rPrP with pathogenic mutations have been used extensively as model systems for studying the conversion processes [5] Some mutant rPrPs have been shown to acquire certain physical characteristics similar to PrPSc, such as the content of b-sheet structure, partial resistance to proteinase K and a propensity to aggregate [6–8] However, the mechanisms leading to these changes are not completely understood Biophysical studies suggest that thermo-instability is not the major contributing factor in the conversion process [9] Accumulated in vivo and in vitro evidence suggest that the conversion process may require the participation of other proteins, such as ‘protein X’ or non-protein macromolecules, such as nucleic acids, glycosaminoglycans, lipids or divalent cations [1,10] Recently, we found that rPrP with a pathogenic mutation of three additional insertions, rPrP8OR, has a more exposed N-terminus, binds better to glycosaminoglycans (GAGs) and is more susceptible to oxidative attack than wild-type rPrP The aberrant properties associated with rPrP8OR are also observed in another insertion mutant prion protein with five extra repeats, rPrP10OR; the aberrations are even more profound in rPrP10OR [11] In addition, we also found that under denaturing conditions and low pH, the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts [12] Here we report further studies on the consequences of binding of GAG and Cu2+ to rPrPs We found that both GAG and Cu2+ promote the aggregation of rPrP in proportion to the number of inserts Furthermore, we found that the octapeptide-repeat region is critical for rPrP aggregation irrespective of whether aggregation is promoted by GAG or Cu2+ Blocking with anti-PrP mAb revealed that GAG and Cu2+ promote the aggregation of rPrP differently Because aggregation is an essential step in PrPC to PrPSc conversion, the significance of these findings with respect to the pathogenesis of inherited human prion disease is discussed Results Enhancement of rPrP aggregation with GAG We previously reported that insertion mutant rPrPs such as rPrP8OR and rPrP10OR bind much better to Aberrant features of insertion mutant prion proteins GAG than rPrP [11] Furthermore, the level of GAG binding is proportional to the number of inserts [11] We also showed that at low pH, for example pH 4.0, rPrPs aggregate spontaneously, again proportional to the number of inserts [12] We therefore investigated whether heparin, a GAG, promotes the aggregation of rPrPs, and whether the degree of enhancement is proportional to the number of inserts These experiments were carried out in NaCl ⁄ Pi at pH 7.4, with low concentrations of rPrPs and GAG; conditions that are more physiological At pH 7.4, heparin enhances the aggregation of all three rPrPs, and the enhancement is greatest for rPrP10OR followed by rPrP8OR and then rPrP (Fig 1) Heparin does not promote the aggregation of rPrPDKKRPK, which lacks the GAG-binding motif, KKRPK, the first five amino acids at the Fig Aggregation of rPrP is enhanced by heparin (A) Comparison of the heparin-enhanced aggregations of rPrP, rPrP8OR, rPrP10OR and rPrPDKKRPK rPrPs (1 lM) were mixed with various concentrations of heparin in NaCl ⁄ Pi (pH 7.4) at 25 °C, and A405 was recorded 300 s after mixing The results are means ± SEM for three experiments (B) Kinetics of the heparin-enhanced aggregations of rPrP, rPrP8OR, rPrP10OR and rPrPDKKRPK rPrPs (1 lM) were mixed with lgỈmL)1 heparin in NaCl ⁄ Pi at 25 °C, and A405 was monitored as described in the Experimental Procedures The enhanced aggregation is given here as an increased percentage of starting turbidities [P = (T ⁄ T0)1) · 100; P, percentage increase; T, turbidity; T0, starting turbidity] All experiments were carried out at least three times with different batches of rPrPs FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS 5565 Aberrant features of insertion mutant prion proteins S Yu et al N-terminus These findings provide the first evidence that enhanced GAG binding has biological consequences on insertion mutant proteins, allowing them to bind GAG better, which then facilitates aggregate formation Because commercially purchased heparin is heterogeneous in its molecular mass, we next investigated whether heparin with a defined molecular mass of kDa, which contains nine sugar residues, also promotes rPrPs aggregation We obtained similar results with this low molecular mass heparin However, heparin with only two sugar residues did not promote aggregation, indicating that a minimal size is required for aggregate promotion (Fig 2A,B) We next used an ELISA to determine whether the aggregates contain GAG A biotinylated GAG was used to promote the aggregation of rPrP or rPrPDKKRPK After aggregation, the rPrP aggregates were collected by repeated centrifugation and washing Aggregates were then resuspended, diluted in various amounts of NaCl ⁄ Pi and added to individual ELISA wells, which had been pre-coated with an anti-PrP mAb, 11G5, to capture the rPrP An avidin-conjugated enzyme was then added to the wells to detect bound biotinylated GAG Much stronger immunoreactivity is detected in samples containing rPrP than rPrPDKKRPK, which cannot bind GAG (Fig 2C) These results suggest that rPrP aggregates indeed contain GAG Sucrose-gradient centrifugation of rPrP–GAG aggregates We used sucrose-gradient centrifugation to compare the relative sizes of rPrP–GAG and PrP10OR–GAG aggregates rPrPDKKRPK was used as a control rPrP– GAG aggregates and controls (without GAG) were centrifuged on 5–50% sucrose gradients Ten fractions from each gradient were collected, run on 12% SDS ⁄ PAGE and immunoblotted with mAb 8H4 Without GAG, rPrP, rPrPDKKRPK and rPrP10OR were detected in the upper fractions (Fig 3) By contrast, when mixed with kDa GAG, rPrP immunoreactivity is detected in all fractions, with the bottom fractions containing most immunoreactivity These results suggest that rPrP–GAG aggregates exist in different sizes By contrast, when rPrP10OR is mixed with kDa GAG, all the immunoreactivity is detected in the bottom fraction Therefore, rPrP10OR forms much larger aggregates than wild-type rPrP In rPrPDKKRPK, which does not bind GAG, when mixed with GAG and centrifuged under identical conditions, all the immunoreactivity remained on the top of the gradient 5566 Fig Characterization of the heparin enhanced aggregation of rPrP (A) Comparison of the aggregation of rPrP enhanced by heparin, low molecular mass heparin (LMW heparin, kDa) and heparin disaccharide rPrP (5 lM) was mixed with various concentrations of heparin, LMW heparin or heparin disaccharide, respectively in NaCl ⁄ Pi at 25 °C, and the A405 was recorded 300 s after mixing The results are means ± SEM for three experiments (B) Kinetics of the aggregation of rPrP enhanced by heparin, LMW heparin and heparin disaccharide rPrP (5 lM) was mixed with 10 lgỈmL)1 of heparin, LMW heparin or heparin disaccharide respectively in NaCl ⁄ Pi at 25 °C, and the A405 was monitored as described in the text (C) Detection of biotinylated heparin in the aggregates of rPrP rPrP (5 lM) was mixed with 10 lgỈmL)1 biotinylated heparin in NaCl ⁄ Pi and the aggregates were harvested by centrifugation at 13 000 g for 10 The pellet was washed with NaCl ⁄ Pi three times and dissolved in NaCl ⁄ Pi containing 0.1% Triton X-100 as described in the text Various dilutions of the resolved aggregate solution were incubated with mAb 11G5 pre-coated plates and the biotinylated heparin, which bound in the aggregates was detected using horseradish peroxidase–streptavidin FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS S Yu et al Fig Sucrose-gradient centrifugation of rPrP–GAG aggregates rPrP (1 lM) was mixed with lgỈmL)1 low molecular mass heparin (3 kDa) in NaCl ⁄ Pi and incubated at 25 °C for 30 The mixture was loaded on to a 5–50% sucrose gradient and centrifuged at °C, 100 000 g for h Ten fractions were drawn from top to bottom An equal volume of each fraction was loaded onto a 12% SDS ⁄ PAGE and PrPs were detected by immunoblotting with mAb 8H4 Enhancement of rPrP aggregation by Cu2+ or Zn2+ but not Mg2+ or Mn2+ rPrP binds divalent cations such as Cu2+ and Zn2+ [13,14] We next determined whether Cu2+ or Zn2+ influences the aggregation of rPrP, rPrP8OR and Aberrant features of insertion mutant prion proteins rPrP10OR At low pH, neither Cu2+ nor Zn2+ has any effect on the aggregation of rPrP (not shown) The failure of these cations to modulate rPrP aggregation is most likely due to the effects of pH on the octapeptide repeat, rendering it unable to bind divalent cations [15] However, when the aggregation assay was carried out at pH 7.4, Cu2+ and Zn2+, but not Mg2+ or Mn2+, promote rPrP aggregation in a concentrationdependent manner (Fig 4A–D) Again, the levels of enhancement are proportional to the number of inserts These results are in good accord with earlier findings that PrP binds Cu2+ and Zn2+ but not Mg2+ or Mn2+ [13,16] Furthermore, although the KKRPK deletion mutant was totally unable to form aggregates in the presence of heparin, in the presence of Cu2+, the KKRPK deletion mutant behaved identically to wild-type rPrP (Fig 5A,B) In addition to the octapeptide-repeat region, two additional Cu2+-binding sites have been identified in the rPrP C-terminal globular domain [16,17] To investigate whether the octapeptide-repeat region is important in Cu2+-induced aggregation, we deleted the octapeptide-repeat region and created rPrPD51-90 In contrast to wild-type rPrP, Cu2+ does not promote the aggregation of rPrPD51-90 (Fig 5C,D) Therefore, the octapeptide-repeat region is the critical motif that mediates Cu2+-induced rPrP aggregation Unexpectedly, GAG also failed to promote the aggregation of rPrPD51-90 (Fig 6A) This deficit is not because rPrPD51-90 does not bind GAG rPrPD51-90 does bind GAG albeit with lower avidity (Fig 6B) Fig Aggregation of rPrPs is enhanced by metal ions One micromole rPrP, rPrP8OR or rPrP10OR was mixed with various concentrations of CuCl2 (A), ZnCl2 (B), MnCl2 (C) and MgCl2 (D) respectively in NaCl ⁄ Pi, and A405 was recorded 300 s after mixing The results are means ± SEM of at least three experiments All the enhanced aggregation are given here as an increased percentage of starting turbidities [P = (T ⁄ T0)1) · 100; P, percentage increase; T, turbidity; T0, starting turbidity] FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS 5567 Aberrant features of insertion mutant prion proteins S Yu et al Fig Copper enhances aggregation of rPrP and rPrPDKKRPK, but not rPrPD51-90 Various dilutions of CuCl2 were mixed with rPrP (A), rPrPDKKRPK (B) and rPrPD51-90 (C), respectively in NaCl ⁄ Pi and A405 was recorded 300 s after mixing (D) A comparison of the aggregation of rPrP, rPrPDKKRPK and rPrPD51-90 enhanced by 50 lM CuCl2 in NaCl ⁄ Pi All the enhanced aggregation are given here as an increased percentage of starting turbidities [P = (T ⁄ T0)1) · 100; P, percentage increase; T, turbidity; T0, starting turbidity] And the results are means ± SEM for at least three experiments At higher protein concentrations, rPrPD51-90 and rPrP have comparable GAG-binding activity (Fig 6B) These results suggest that the octapeptide-repeat region is the nucleation center of rPrP aggregation, irrespective of whether aggregation is initiated with GAG or Cu2+ Furthermore, these results also provide strong evidence that although the KKRPK motif is the GAG-binding site, the octapeptide-repeat region also contributes to the total affinity between PrP and GAG Although Cu2+ and GAG bind to different sites on PrP, we did not observe a synergistic effect when both Cu2+ and GAG were added to the rPrPs (results not shown) Sucrose-gradient centrifugation of rPrP–Cu2+ aggregates We also used sucrose-gradient centrifugation to compare the relative sizes of rPrP–Cu2+ and PrP10OR– Cu2+ aggregates rPrPD51-90 was used as a control As expected, without Cu2+, rPrP, rPrPD51-90 and rPrP10OR were detected in the top fractions (Fig 7) By contrast, when rPrP is mixed with Cu2+, most of the PrP immunoreactivity is detected in the bottom fractions However, upon longer exposure, PrP immunoreactivity is also present in the intermediate fractions (not shown) By contrast, when rPrP10OR is mixed with Cu2+, all the immunoreactivity is detected in the bottom fraction Therefore, rPrP10OR also forms much larger aggregates than wild-type rPrP rPrPD51-90, which does not bind Cu2+, remained on the top of the gradient 5568 Modulation of GAG- or Cu2+-promoted aggregation of rPrP10OR with anti-PrP mAbs We next investigated whether GAG- or Cu2+-promoted aggregation of rPrP10OR can be amended with anti-PrP mAbs The epitopes of these mAbs are diagrammatically presented in Fig 8A Of all the anti-PrP mAbs tested, one, 8B4, consistently enhanced the aggregation of rPrP10OR in the presence of either GAG or Cu2+ (Fig 8B,C) mAb 8B4 alone does not induce the aggregation of rPrP10OR without the PrP ligands Four mAbs, SAF32, 11G5, 7A12 and 8H4 consistently blocked the aggregation of rPrP10OR in the presence of GAG (Fig 8B) However, none of the tested mAb was able to block the effects of Cu2+ (Fig 8C) The inability of these mAbs to block Cu2+ induced aggregation is not because Cu2+ prevents the binding of these mAbs as shown by ELISA; Cu2+ does not inhibit the binding of these mAbs to rPrP (results not shown) Discussion Aggregation of PrP is an essential step in the conversion of PrP to PrPSc [1] Here we describe four new findings on the aggregation of rPrPs: (a) in the presence of PrP ligands, such as GAG or the divalent cation Cu2+, rPrPs aggregate in proportion to the number of octapeptide inserts, thus rPrPs with insertional mutations, such as rPrP8OR and rPrP10OR form more and larger aggregates with faster kinetics than wild-type rPrP; (b) whereas GAG-induced aggregation FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS S Yu et al Fig Heparin enhances aggregation of rPrP, but not rPrPDKKRPK and rPrPD51-90 (A) rPrPs (1, 3, lM) were mixed with lgỈmL)1 heparin in NaCl ⁄ Pi and A405 was measured 300 s after mixing All the enhanced aggregation are given here as an increased percentage of starting turbidities [P = (T ⁄ T0)1) · 100; P, percentage increase; T, turbidity; T0, starting turbidity] The results herein are means ± SEM for three experiments (B) Detection of rPrPD51-90 binding to heparin by ELISA Heparin (10 lgỈmL)1) was coated onto plates at °C overnight and blocked with 3% BSA BSA was coated as a control Different concentrations of rPrP, rPrPDKKRPK or rPrPD51-90 were incubated with the plates for h at 25 °C After three washes with PBST, appropriate dilution of mAb 8H4 was used to detect the bound rPrP The results are means ± SEM for three wells and this experiment was repeated at least three times requires the GAG-binding motif, Cu2+-induced aggregation requires the octapeptide repeat; (c) the octapeptide-repeat region is essential for both GAG- and Cu2+-promoted rPrP aggregation; (d) aggregation induced by GAG and Cu2+ share common features, yet each one has its own unique features, suggesting multiple pathways leading to rPrP aggregation Bacterial produced rPrP has been used extensively as a model system for studying the aggregation process [5] In previous studies, aggregation of rPrP required denaturation, low pH and relatively high concentrations of rPrP [18–22] In this study, aggregation of rPrP was carried out at pH 7.4 and with relatively low concentrations of full-length rPrP; these conditions are physiologically more relevant Accumulated evidence suggests that binding of GAG may be important in the pathogenesis of prion diseases [23–27] PrPSc parti- Aberrant features of insertion mutant prion proteins Fig Sucrose-gradient centrifugation of rPrP–Cu2+ aggregates rPrPs (1 lM) was mixed with 20 lM CuCl2 in NaCl ⁄ Pi and incubated at 25 °C for 30 The mixture was loaded on top of a 5–50% sucrose gradient and centrifuged at °C, 100 000 g for h Ten fractions were drawn from top to bottom An equal volume of each fraction was loaded onto 12% SDS ⁄ PAGE and the PrPs were detected by immunoblotting with mAb 8H4 cles formed in vivo contain GAG [28] In vitro, GAG facilitates the conversion of PrP to PrPSc [24], and greatly increases the infectivity of non-aggregated PrPres [25] Reduction of cellular GAG significantly decreases the biogenesis of PrPSc in scrapie-infected cells [29] Cell-surface GAG has also been reported to be the receptor for PrPSc [23,27] However, exogenous GAG and GAG analogs, such as low molecular mass heparin, suramin, pentosan polysulfate and dextran sulfate can inhibit PrPSc formation in cells, and prolong the incubation time of experimental prion diseases [10] It has been postulated that exogenous GAG and GAG analogs block PrPSc formation by competing with the endogenous GAG which is critical for PrPSc generation [10] GAG may function as a scaffold for concentrating PrP, creating a reservoir of PrP for conversion We reported earlier that rPrP8OR and rPrP10OR bind GAG better than rPrP, and the level of binding is proportional to the number of inserts [11] Our current findings that GAG also promotes the aggregation of rPrP8OR and rPrP10OR proportional to the number of inserts are in good accord with our earlier results Enhancement of rPrP aggregation is most apparent when the concentration of rPrP is low, such as lm At this concentration, rPrP by itself does not aggregate A small GAG, with nine sugar residues is as FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS 5569 Aberrant features of insertion mutant prion proteins S Yu et al Fig Blocking of rPrP10OR aggregation enhanced by heparin or copper using antiPrP mAbs (A) The location of mAb-binding epitopes along the length of PrP LS, leader sequence; GPI, glycosylphosphatidylinositol anchor (B) Blocking of the aggregation of rPrP10OR enhanced by heparin rPrP10OR (1 lM) was mixed with lgỈmL)1 heparin and 0.125 lM mAbs in NaCl ⁄ Pi and A405 was monitored as described in text NS mAb, non-specific mAb (C) Blocking of the aggregation of rPrP10OR enhanced by copper rPrP10OR (1 lM) was mixed with 20 lM CuCl2 and 0.125 lM mAbs in NaCl ⁄ Pi and A405 was recorded The aggregation is given as an increased percentage of starting turbidities [P = (T ⁄ T0)1) · 100; P, percentage increase; T, turbidity; T0, starting turbidity] The two experiments were repeated at least three times effective as larger GAG in promoting rPrP aggregation However, a disaccharide of GAG is unable to cause aggregation, suggesting that the minimum unit of GAG required for rPrP aggregation is between three and nine sugar residues The promotion of aggregation by GAG is not only limited to rPrP with insertion mutations GAG also promotes the aggregation of rPrPs with pathogenic point mutations, albeit at lower levels [30] We hypothesize that enhanced binding to GAG, leading to aggregation is a common feature in inherited human prion disease The precise mechanism by which GAG promotes rPrP aggregation is not known GAG may promote aggregation by serving as a scaffold If this is the case, the rPrP aggregates should contain GAG Alternatively, GAG may simply serve as a platform for rPrPs to be physically close to each other, resulting in aggregation between rPrPs, without including GAG Our ELISA results suggest that some rPrP–GAG aggregates contain GAG However, our sucrose-gradient centrifugation experiments revealed that rPrP–GAG 5570 aggregates exist in many different sizes Because the GAG used in these experiments has a molecular mass of kDa, it is probable that some of the larger rPrP– GAG aggregates are composed mainly of rPrP Thus, GAG serves as a scaffold as well as a platform in facilitating rPrP aggregation In contrast to rPrP, when mixed with kDa GAG, all the rPrP10OR is detected in the bottom fraction of the sucrose gradient This is in good accordance with our earlier finding that under denaturing and low pH condition; rPrP10OR has the propensity to spontaneously aggregate, in a protein concentration-dependent manner When incubated with GAG, rPrP10OR is concentrated, thus able to form much larger aggregates It is interesting to note that in PrPSc infected mouse brain homogenate centrifuged under identical conditions, most of the PrP immunoreactivity is present in the bottom fractions of the sucrose gradient [31] However, in contrast to in vivo-derived PrPSc aggregates, the rPrP aggregates formed in the presence of GAG are PK sensitive (results not shown) FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS S Yu et al rPrP binds divalent cations, such as Cu2+ and Zn2+ but not Mg2+ or Mn2+ [32] A metal imbalance in the central nervous system has been speculated to play a role in neurodegenerative diseases, including prion disease [32] However, the physiological significance of the interaction between PrP and Cu2+ remains poorly understood Some studies found that Cu2+ causes aggregation of rPrP [33–35] Others reported that Cu2+ inhibits rPrP conversion to amyloid [36,37] Copper chelators also inhibit PrPSc replication in vitro [38] Some studies suggest that treatment with Cu2+ causes PrP to acquire PK resistance [34–36,39,40] However, this interpretation is complicated by the recent finding that Cu2+ inhibits proteinase K activity [41] We found that at neutral pH and low concentrations of rPrPs, Cu2+ and Zn2+ but not Mg2+ and Mn2+ promote aggregation of rPrP, rPrP8OR and rPrP10OR in a concentration-dependent manner For rPrP8OR and rPrP10OR, the enhancement can be observed in as low as lm of Cu2+ or Zn2+, a concentration that is physiologically relevant [42] Again the degree of enhancement is proportional to the number of octapeptide repeats, and Cu2+ is consistently more efficient in promoting aggregation than Zn2+ Cu2+ does not promote the aggregation of rPrPD51-90, which lacks the octapeptide-repeat region Therefore, the octapeptiderepeat region is important in rPrP aggregation This finding is consistent with an earlier report suggesting that the octapeptide-repeat region constitutes a pHdependent folding and aggregation site of PrP [22] Our result is also consistent with another study showing that when Cu2+ binds to the octapeptide-repeat region, it serves as a ‘copper switch’, which is important in PrP aggregation [43] However, we were surprised to find that GAG was also unable to promote the aggregation of rPrPD51-90, because the octapeptiderepeat region is not required for the binding of GAG Furthermore, under high rPrPD51-90 concentration, low pH and denaturing conditions, rPrPD51-90 also failed to aggregate spontaneously (results not shown) Therefore, the octapeptide-repeat region is critical for rPrP aggregation irrespective of whether aggregation is ligand initiated or spontaneous It should be noted that others have identified additional Cu2+-binding sites at the C-terminus of PrP [16] It is possible that these binding sites may not be essential for PrP aggregation The precise mechanisms by which the divalent cations promote aggregation are not known Cu2+ and Zn2+can bind PrP intramolecularly as well as intermolecularly [44] We speculate that it is the intermolecular binding of Cu2+ that enhances aggregation Aberrant features of insertion mutant prion proteins Presumably, by having more octapeptide repeats, rPrP10OR is more readily to interact with Cu2+ and Zn2+ The failure of either Mg2+ or Mn2+ to enhance aggregation provides the most appropriate control for the specificity of the interactions This interpretation is also supported by results from the sucrose gradient centrifugation experiments It has been reported that GAG promotes the aggregation of rPrP and that the aggregate is stabilized by the binding of Cu2+ [26] However, we did not observe a synergistic effect between GAG and Cu2+ in our aggregation assay (results not shown) It should be noted that in our assay the concentrations of rPrPs (1 versus lm), GAG (0.1 versus lm) as well as Cu2+ (1–20 versus 500 lm) were much lower than is typically used in this type of experiments Furthermore, our assay only detects the amount of aggregate that is generated rather than the stability of the aggregate Hence, it is possible that the aggregate formed with GAG alone is different from the aggregate formed in the presence of high concentrations of GAG and Cu2+ We reported earlier that under low pH and denaturing conditions, only mAbs which react with an epitope in the octapeptide-repeat region and the helix region respectively, block the spontaneous aggregation of rPrPs [12] In the current study, we found that mAb 8B4, which reacts with an epitope at the N-terminus further promotes GAG- and Cu2+-induced rPrP aggregation We suggest that mAb 8B4 is able to align the rPrP in the same orientation, in parallel, pairing the N-terminus of two PrPs, which then facilitates the binding of either GAG or Cu2+ It should be noted that mAb 8B4 does not cause the aggregation of rPrP without the participation of either GAG or Cu2+ In addition to mAbs that are specific for the octapeptide-repeat region, such as SAF32, other mAbs, such as 7A12, 11G5 and 8H4 also blocked GAGinduced aggregation These results suggest that the entire C-globular domain including the helix 1, b2 and helix regions are all important in the aggregation process We could not evaluate whether mAb 8H4 inhibits spontaneous aggregation of rPrP because mAb 8H4 does not bind PrP at pH 4.0 This observation is in good accordance with a recent study suggesting that the opening of the helix region, followed by conformational changes in helix of rPrP, is critical in rPrP aggregation [45] Finally, we showed that mAb 8F9, which reacts with an epitope at the C-terminal end, does not block GAG-induced aggregation These results suggest that in the presence of GAG, aggregation of rPrP starts at the end of N-terminus, proceeding into the octapeptide-repeat region, the b1-sheet FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS 5571 Aberrant features of insertion mutant prion proteins S Yu et al region, helix region and then the helix 2, in a ‘zipper’-like manner This interpretation is also in good agreement with another recent finding showing that PrP fibril formation proceeds by aligning PrP molecules in parallel, face to back, like a ‘zipper’ [46] The underlying reason that none of the anti-PrP mAbs is able to block Cu2+-induced rPrP aggregation is not known Accumulated evidence suggests that there are multiple pathways in the PrP aggregation process [10,47] Our results suggest that GAG-induced and Cu2+-induced aggregation proceed via different pathways All the studies described here were based on findings using rPrPs Normal PrP has two highly conserved N-linked glycosylation sites and is present on the cell membrane with a glycosylphosphatidylinositol anchor Therefore, it is possible that the presence of N-linked glycans as well as the placement of the cell membrane can further modulate the interactions between PrP and its ligands Based on our findings, we hypothesize that an increase in the number of octapeptide repeats causes conformational changes at the N-terminus, resulting in an enhancement in the binding of PrP ligands, such as GAG, eventually leading to PrP aggregation Because all these aberrant features are proportional to the number of insertions, our earlier and current findings provide a biochemical explanation for the observation that patients with more octapeptiderepeat insertions have earlier disease onset and shorter disease duration [3,48] Experimental procedures Plasmid construction and recombinant protein preparation Cloning, generation and purification of human rPrP, rPrP8OR, rPrP10OR and rPrPDKKRPK were performed as described previously with slight modification [11,30] After refolding and purification, these rPrPs were dialyzed against 20 mm NaAc, pH 5.5 and filtered through a 0.2 lm membrane For human rPrPD51-90, codons 51–90 were removed from the prion protein coding sequence by annealing the primer 5¢-GGCAACCGCTACCCA ⁄ CAAGGAGGTGG CACC-3¢ ( ⁄ marks the site between codon 50 and codon 91) to a phagemid containing the PrP-coding sequence Mutagenesis was performed using the BioRad Muta-Gene phagemid in vitro mutagenesis kit The PrP mature fragment (codons 23–231 with deletion of residues 51–90) was cloned to the vector of pET42a(+) (Novagen, Gibbstown, NJ, USA) [11], termed pET–rPrPD51-90 The insertion sequence was verified by using the Applied Biosystems 3730 sequencer (Foster City, CA, USA) 5572 Freshly transformed BL21 (DE3) star Escherichia coli (Invitrogen, Carlsbad, CA, USA) containing plasmid pET– rPrPD51-90 was transferred to L Luria–Bertani media with 50 lgỈmL)1 kanamycin at 37 °C until A600 reached 0.6 and induced for h with mm isopropyl thio-b-d-galactoside Bacteria were harvested by centrifugation at 4000 g for 15 at °C, resuspended in 20 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl, mm phenylmethanesulfonyl fluoride, 0.1 mgỈmL)1 lysozyme, mm EDTA, 0.1% Triton X-100 and incubated at 25 °C for 30 before further lysis by sonication Samples were centrifuged at 13 000 g for 15 min, and the protein pellets were extensively washed using 20 mm Tris ⁄ HCl, pH 7.4 with 0.5% Triton X-100 twice, then washed with the same buffer containing m NaCl and m urea respectively The pellets were then resuspended in 20 mm Tris ⁄ HCl, pH 8.0, m urea, 10 mm b-mercaptoethanol The protein was refolded by dialysis against 20 mm Tris ⁄ HCl, pH 8.0 buffer with decreasing urea and b-mercaptoethanol gradient concentrations All refolded rPrPs were further dialyzed against 20 mm NaAc, pH 5.5, filtered through 0.2 lm membrane, stored at )80 °C and used for experiments within one week after refolding SDS ⁄ PAGE and Coomassie Brilliant Blue staining showed that the purity of the recombinant protein is consistently > 95% (not shown) Protein concentration was determined with a Bio-Rad Protein Assay Kit All of the recombinant prion proteins were freshly purified before use Antibodies The generation, purification and characterization of all the anti-PrP murine mAbs have been described in detail previously [49,50] mAb 8B4 recognizes an epitope at residues 35–45; SAF32 reacts with residues 63–94 covering the octapeptide-repeat sequences [51]; 7A12 interacts with helix between residues 143 and 155; 11G5 reacts with residues 115–130 covering b-sheet 1; 8H4 recognizes residues 175– 185 of helix 2; 8F9 reacts with residues 220–231 mAbs 8B4, SAF32, 7A12, 8H4 and 8F9 are IgG1, whereas mAb 11G5 is IgG2b All mAbs were affinity purified using Protein G chromatography The concentration of mAbs was determined with a BCA protein assay Kit (Pierce, Rockford, IL, USA) Turbidity measurement The assays were performed at 25 °C in flat-bottomed 96-well plates Heparin (from porcine intestinal mucosa; Sigma, St Louis, MO, USA) or CuCl2 was added into the wells before addition of 200 lL NaCl ⁄ Pi (pH 7.4) containing lm rPrPs After mixing as quickly as possible, turbidities were monitored within 15 s by reading the absorbance at 405 nm in a Beckman Coulter AD340 micro-ELISA FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS S Yu et al plate reader, using a kinetic photometric model (interval time 30 s, 30 cycles with s shaking before every cycle) Similar processes were performed with ZnCl2, MgCl2 and MnCl2 To investigate whether anti-PrP mAbs can block the heparin enhanced aggregation of rPrP, 10 lL heparin (final concentration lgỈmL)1) was mixed with 2.5 lL mAbs (final concentration 0.125 lm) Then 200 lL NaCl ⁄ Pi containing lm rPrP10OR was added and mixed quickly Turbidities were recorded as described in above A similar procedure was carried out to investigate the effect of anti-PrP mAbs on copper enhanced aggregation of rPrP An irrelevant mAb 9C1, anti-(brain-derived neurotrophic factor), was used as a negative control All experiments were carried out at least three times with different batches of rPrPs Detection of rPrP binding to heparin Flat-bottomed, 96-well Costar plates (Corning, Corning, NY, USA) were coated with 10 lgỈmL)1 heparin at °C overnight and blocked with 3% BSA in NaCl ⁄ Pi at 25 °C for h BSA was coated onto the plates as a control Appropriate dilutions of rPrPD51-90 or rPrP were added into the plates in triplicate and incubated at 25 °C for h After three washes with phosphate-buffered saline ⁄ 0.05% Tween (PBST), bound rPrP was detected with mAb 8H4 Horseradish peroxidase-conjugated goat anti-mouse IgG (Chemicon, Billerica, MA, USA) was used as the secondary antibody and A405 was measured for 2,2¢-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN, USA) All experiments were carried out at least three times with different batches of rPrPs Aberrant features of insertion mutant prion proteins heparin was detected by adding horseradish peroxidase-conjugated streptavidin (Chemicon) at : 10 000 dilutions 2,2¢Azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) was added and A405 was recorded All experiments were carried out at least three times with different batches of rPrPs Sucrose-gradient fractionation To form a 5–50% step sucrose gradient, 5, 10, 15, 20, 30, 40, 50% sucrose solution prepared in NaCl ⁄ Pi were loaded into ultraclear centrifuge tubes (13 · 51 mm) rPrPs (1 lm) were mixed with lgỈmL)1 low molecular mass heparin (3 kDa) or 20 lm CuCl2 in NaCl ⁄ Pi After incubation for 30 at 25 °C, 0.5 mL of the mixture was loaded on top of the sucrose gradient Ultracentrifugation was carried out in SW55 rotor (Beckman, Fullerton, CA, USA) at 100 000 g, °C for h Fractions of 0.5 mL were collected from the top of the tubes rPrP present in different sucrosegradient fractions was detected by immunoblotting 10 lL of each fraction was mixed with 2· SDS loading buffer and heated at 95 °C for 10 before separation on 12% SDS ⁄ PAGE The gel was transferred to a nitrocellulose membrane and probed with mAb 8H4 Blue dextran (Sigma) with a molecular mass of 2000 kDa was used as a marker in the gradient Statistical analysis A two-way ANOVA program was used to determine the P-value between various groups P > 0.05 is considered to be not significant (ns) Acknowledgements Detection of biotinylated heparin in the aggregates of rPrPs mAb 11G5 was previously shown to be able to react with PrP aggregates [52] mAb 11G5 was coated onto the flatbottomed, 96-well Costar plates at lgỈmL)1 at °C overnight and blocked with 3% BSA in NaCl ⁄ Pi at 25 °C for h BSA was coated onto the plates as a control Five micromoles of either rPrP or rPrPDKKRPK was mixed with 10 lgỈmL)1 biotinylated heparin (from porcine intestinal mucosa, Sigma) in 400 lL NaCl ⁄ Pi respectively and incubated at 25 °C for 30 The aggregates were collected by centrifugation at 16 000 g for 10 at 25 °C Supernatants were removed and the pellets were washed three times with NaCl ⁄ Pi by vortexing followed by centrifugation at 16 000 g for The aggregates were dissolved with 50 lL NaCl ⁄ Pi containing 0.1% Triton X-100 by incubation at 42 °C for 10 NaCl ⁄ Pi (450 lL) was then added into the Eppendorf tubes to a final volume of 500 lL Various dilutions of this original aggregate solution in NaCl ⁄ Pi were then incubated with mAb 11G5-coated plates at °C overnight After three washes with PBST, the bound biotinylated We would like to thank Dr Jacques 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dimers and PrP(Sc) aggregates J Virol 79, 12355–12364 FEBS Journal 275 (2008) 5564–5575 ª 2008 The Authors Journal compilation ª 2008 FEBS 5575 ... proportional to the number of inserts [12] We therefore investigated whether heparin, a GAG, promotes the aggregation of rPrPs, and whether the degree of enhancement is proportional to the number of inserts... conditions, all the immunoreactivity remained on the top of the gradient 5566 Fig Characterization of the heparin enhanced aggregation of rPrP (A) Comparison of the aggregation of rPrP enhanced... induce the aggregation of rPrP10OR without the PrP ligands Four mAbs, SAF32, 11G5, 7A12 and 8H4 consistently blocked the aggregation of rPrP10OR in the presence of GAG (Fig 8B) However, none of the