Plasminogen

Abstract

The biochemical essence of prion replication is the molecular multiplication of the disease-associated misfolded isoform of prion protein (PrP), termed PrPSc, in a nucleic acid-free manner. PrP^Sc is generated by the protein misfolding process facilitated by conformational conversion of the host-encoded cellular PrP to PrP^Sc. Evidence suggests that an auxiliary factor may play a role in PrP^Sc propagation. We and others previously discovered that plasminogen interacts with PrP, while its functional role for PrPSc propagation remained undetermined. In our recent in vitro PrP conversion study, we showed that plasminogen substantially stimulates PrP^Sc propagation in a concentration-dependent manner by accelerating the rate of PrP^Sc generation, while depletion of plasminogen, destabilization of its structure, and interference with the PrP-plasminogen interaction hinder PrP^Sc propagation. Further investigation in cell culture models confirmed an increase of PrP^Sc formation by plasminogen. Although molecular basis of the observed activity for plasminogen remain to be addressed, our results demonstrate that plasminogen is the first cellular protein auxiliary factor proven to stimulate PrP^Sc propagation.

Prions are unique infectious particles that replicate in the absence of nucleic acids1 and cause fatal neurologic disorders in mammals.2 In fact, the prion particle is composed of an alternatively folded form of the cellular prion protein (PrP^C) encoded by the Prnp gene. The misfolded form of PrP^C, referred to as PrP^Sc, shares the same primary structure with PrP^C,3 but exhibits distinctively different biochemical and biophysical properties.4,5 Moreover, animals lacking expression of the Prnp gene are resistant to prion infection,6 suggesting that PrP^C serves as a precursor for PrP^Sc.

The essence of prion biosynthesis is based on the protein-only hypothesis that postulates self-perpetuating replication of the prion protein.1 Although the exact replication process remains elusive, the template-assisted conversion model proposes an idea that PrP^Sc serves as a template to convert α-helices of PrP^C into the β-sheets of PrP^Sc during prion replication.7 According to many lines of evidence, there exists an unidentified auxiliary factor, designated protein X, which favorably interacts with PrP^C to produce a thermodynamically stable intermediate conformation called PrP*.8 By introducing PrP^Sc to a host, dimers will readily form between PrP* and PrP^Sc. This interaction induces PrP* to take on the conformation of PrP^Sc and results in a complex consisting of the template and the newly formed PrP^Sc molecules. Once the dimer disassociates protein X and the two PrP^Sc molecules are released and allowed to continue replicating in an exponential fashion. Recent observations that infectious material can be generated in vitro using recombinant PrP have authenticated the protein-only hypothesis.9,10 However, the infectivity of these in vitro generated PrP^Sc products is lower than that of brain-derived PrP^Sc, leading to the possibility that insufficient levels of the unidentified auxiliary factor are the limiting factor for these in vitro assays.

To date, non-mammalian chaperone proteins, sulfated glycans and certain polyanionic macromolecules, such as RNA, have shown to increase the level of PrP^Sc in several different in vitro assays.11–14 However, defining these molecules as cellular auxiliary factors that promote the conversion of PrP^C to PrP^Sc has been prevented for several reasons. First, overexpression of yeast heat shock protein Hsp 104 in transgenic mice does not modulate the incubation time of disease and PrP^Sc accumulation upon prion inoculation.15 Although mammalian Hsp 70 is upregulated in humans and animals with prion diseases,16,17 it participates in downregulation, but not upregulation, of PrP^Sc accumulation.18 Second, the effect of sulfated glycans on PrP^Sc formation has been inconsistent11–13 and certain sulfated glycans inhibit PrP^Sc propagation in animals and cultured cells.19–21 Lastly, although RNA is able to increase PrP^Sc propagation and induce the formation of PrP^Sc de novo from purified PrP^C in the absence of a PrP^Sc seed,22 its specificity is in question. Thus, an auxiliary factor that positively assists PrP^Sc replication and is composed of a mammalian cellular protein remains to be identified.

Our approach to identify an auxiliary factor relies on the idea that the auxiliary factor interacts with PrP^C as proposed in the protein X hypothesis.7 Therefore, we considered PrP ligands as the best candidates for this unidentified factor. By screening a phage display cDNA expression library from ScN2a cells, we identified kringle domains of plasminogen that interact with recombinant PrP folded in an α-helical conformation (α-PrP).23 In vitro binding assays showed that interaction between plasminogen and α-PrP that represents the conformational state of PrP^C was enhanced by the introduction of a dominant negative mutation and the presence of the basic N-terminal sequences in PrP.23 Interaction of PrP^C and plasminogen was further confirmed by the ability of plasminogen and its kringle domains to readily interact with α-PrP.24–29 However, despite greater interaction with α-PrP, plasminogen also interacted with PrP in β-sheet conformations.23 Prior to our study, it was shown that PrP^Sc was immunoprecipitated with beads linked to plasminogen, its first three kringle domains [K(1+2+3)], and more recently the repeating YRG motif found within the plasminogen kringle domains.30–33 However, the ability of plasminogen to bind to PrP^Sc was dependent on conditions of the lipid rafts and plasminogen was actually associated with PrP^C in the intact lipid rafts.34

To determine the functional relevance of this interaction for PrP^Sc replication, we explored whether plasminogen enhances PrP^Sc propagation using cell culture models and the in vitro PrP conversion assay, termed protein misfolding cyclic amplification (PMCA).35 The addition of plasminogen in PMCA resulted in the generation of significantly more PrP^Sc in a concentration-dependent manner (Fig. 1A), suggesting that plasminogen stimulates the conversion of PrP^C to PrP^Sc. Indeed, our kinetic studies showed that plasminogen accelerates the rate of PrP^Sc generation during the early stages of the in vitro PrP conversion reaction (reviewed in ref. 35). In contrast, the addition of plasminogen in PMCA lacking either PrP^C or PrP^Sc failed to generate PrP^Sc (Fig. 1B and C). These results suggest that both PrP^C and PrP^Sc are required for PrP^Sc replication stimulated by plasminogen and that plasminogen facilitates neither spontaneous PrP conversion nor PrP^Sc augmentation through aggregating pre-existing PrP^Sc. Incubation of plasminogen with pre-formed PrP^Sc followed by treatments with proteinase K failed to either increase or decrease the PrP^Sc level compared to controls (Fig. 1D and E), suggesting that plasminogen is not involved in stabilization of PrP^Sc, enhancement of PrP^Sc resistance to protease or promotion of PrP^Sc binding to the membrane for western blotting. The activity to stimulate PrP conversion was a specific property of plasminogen and was not shared with other proteins such as a known PrP ligand and proteins abundantly found in the serum or at the extracellular matrices where plasminogen is present (reviewed in ref. 35). In addition, we found that the ability of plasminogen to assist in PrP^Sc propagation is preserved in its kringle domains (reviewed in ref. 35). Furthermore, the activity associated with plasminogen under cell-free conditions was reproduced in cell culture models. Plasminogen and its kringle domains increased PrP^Sc propagation in cultured cells chronically infected with mouse-adapted scrapie or chronic wasting disease prions (Fig. 2A–D). This suggests that the activity of plasminogen in PrP^Sc replication has biological relevance.

Corresponding to the results that plasminogen positively assists in PrP^Sc replication by stimulating conversion of PrP^C to PrP^Sc, depletion of plasminogen from the PMCA reaction by using brain material derived from plasminogen-deficient mice restricted PrP^Sc replication to the basal level (Fig. 1F). Supplementation with plasminogen for PMCA using plasminogen-deficient brain homogenate restored PrP^Sc propagation to levels equivalent to that of control PMCA in which only brain material of their non-genetically altered littermates was used (Fig. 1G). Furthermore, structural destabilization of plasminogen affected the activity of plasminogen that enhances PrP^Sc propagation. Because intact disulfide bonds are critical in maintaining structural integrity and the binding activity of plasminogen, we conducted PMCA supplemented with either structurally intact or modified plasminogen to investigate the functionality of plasminogen. The result showed that plasminogen pre-treated sequentially with chemical agents that disrupt disulfide bonds and modify free sulfhydryl groups failed to stimulate PrP^Sc propagation (reviewed in ref. 35). In addition, we showed that interference with the plasminogen-PrP interaction using L-lysine abolished the plasminogen-mediated stimulation of PrP^Sc propagation in PMCA (reviewed in ref. 35). Because previous observations in immunoprecipitation30 and ELISA binding assays23 described that L-lysine specifically inhibited formation of the PrP-plasminogen complex, the presence of L-lysine in PMCA is considered to saturate the lysine binding motifs of kringle domains, which competitively prevents the kringle domains of plasminogen from interacting with PrP and inhibited PrP conversion. These various inhibition studies of PrP^Sc propagation provides confirmatory evidence that plasminogen plays an important role in PrP^Sc replication.

Despite our progress in understanding the role of plasminogen in PrP^Sc propagation, we are still unable to address mechanistic details by which plasminogen exerts its function. In fact, plasminogen shares a number of the expected characteristics of the previously proposed auxiliary factor, although there are minor but distinctive discrepancies in their properties (summarized in Table 1). With the insight obtained from known properties of plasminogen, it is possible to postulate a few plausible mechanisms how plasminogen promotes PrP^Sc replication (Fig. 3). First, plasminogen may control conformational rearrangement of PrP^C to PrP*, resembling a molecular chaperone. This scenario is identical to the protein X hypothesis, in which plasminogen replaces protein X to assist the conversion process. Second, plasminogen may promote aggregation of PrP^Sc already converted from PrP^C. This will result in efficient formation of PrP^Sc multimers. Third, plasminogen may stabilize the pre-existing PrP^Sc aggregates so that stabilized aggregates are better protected from degradation or processing by the intrinsic clearance mechanism. This will result in increased accumulation and stability of PrP^Sc. In the second and third scenarios, the function of plasminogen is not involved in PrP^C or its conversion process, but limited to the interaction with pre-formed PrP^Sc. Fourth, plasminogen may play a role as a scaffolding molecule that simply brings both PrP^C and PrP^Sc together within a proximity. This will increase the frequency of interaction between PrP^C and PrP^Sc for conversion. This scenario is distinguished from the other three by postulating plasminogen interaction with both isoforms of PrP. In contrast, plasminogen is assumed to interact with only PrP^C or PrP^Sc in other cases. Although accumulating data including our recent studies provide critical pieces of evidence to envision described mechanistic insights, it is still premature to conclude the mechanism involved in plasminogen-mediated stimulation of PrP^Sc propagation.

It is essential to address a few additional issues of PrP^Sc propagation stimulated by plasminogen aside from the mechanistic details. First, it is necessary to confirm the authenticity of PrP^Sc generated in the aid of plasminogen as a bona fide infectious agent. In addition, it would be interesting to compare the structural signatures of PrP^Sc generated in our study and found the prion seeds. This study may provide a clue to establish a structure-infectivity relationship for PrP^Sc. Furthermore, the ability to repeat our PMCA results in a similar assay reconstituted with defined components would clarify whether plasminogen directly contributes to PrP^Sc propagation. Finally, the contribution of plasminogen to the species barrier by controlling the compatibility between the host PrP^C and prion strains should be determined. Collectively, these studies will be useful to identify an intricate regulatory role for plasminogen during PrP^Sc replication and prion transmission.

Plasminogen has shown that it stimulates PrP^Sc propagation in the cell-free assays and cultured cells, while its role in animal models has not been obviously clarified. In opposition to the anticipated outcomes that the course of prion disease in plasminogen-deficient animals would be delayed, intracerebral prion infection of two independent mouse lines deficient in plasminogen resulted in either unchanged or accelerated disease progression when compared to the appropriate wild-type controls.36,37 These results do not support each other and there is no good interpretation to reconcile their incongruence. However, the discrepancy between the expectation and the actual outcomes of the previous studies can be caused by the intrinsic health problems associated with the mouse lines used for the studies,38–40 which raises a question for the credibility of the model system. Some clinical phenotypes of confounding health problems in plasminogen-deficient mice overlap with the typical clinical signs of prion disease in mice. Furthermore, the drastically shortened life expectancy of plasminogen-deficient mice coincides with the incubation periods of mice inoculated with prion strains used in the previous studies. Lastly, the health problems of plasminogen-deficient mice could exacerbate the progression of the course of disease in mice inoculated with prions. Thus, either unchanged or shortened incubation periods of prion-inoculated plasminogen-deficient mice may not solely reflect the effect of plasminogen deficiency related to prion replication. Alternatively, the reason for observed outcomes from the in vivo models may be associated with the presence of functionally redundant proteins for plasminogen even under its absence. Because kringle domains of plasminogen share a well-conserved structure with those of other proteins such as tissue-type plasminogen activator, hepatocyte growth factor and apolipoprotein (a),41 it is possible to postulate that other proteins that contain kringle domains can functionally replace plasminogen. Therefore, an animal model that circumvents the drawbacks associated with the current models is needed to address the relevance of plasminogen in prion replication in vivo.

Based on our study that identified plasminogen as the first cellular protein cofactor for PrP^Sc propagation, we anticipate an intriguing opportunity to develop future diagnosis and therapeutic intervention for prion disease. Previously, the identity of a proposed auxiliary factor was obscure so most approaches either targeted PrP isoforms or were empirical.42 Our study suggests that plasminogen is a novel target to interfere with PrP^Sc replication. Therefore, a variety of strategies that either deplete plasminogen or interfere with the formation of PrP-plasminogen complexes would work for development of a novel therapeutic intervention for prion disease. For instance, RNA interference of plasminogen expression, monoclonal anti-plasminogen antibody to block plasminogen binding to PrP, L-lysine that saturates the binding sites of plasminogen to PrP, and chemical agents that destabilize the structure of plasminogen are potential strategies for this purpose.

In conclusion, plasminogen is a PrP ligand with the ability to stimulate PrP^Sc propagation. Our findings are indispensable in gaining a better understanding of the underlying mechanism for PrP^Sc propagation, while unveiling a new therapeutic target for prion disease.

Abbreviations

PrP	=	prion protein
PrPSc	=	disease-associated prion protein
PrPC	=	cellular prion protein
Prnp	=	the gene that encodes PrPC
protein X	=	unidentified auxiliary factor for PrP conversion
PrP*	=	an intermediate conformation of PrP generated from PrPC
K(1+2+3)	=	the first three kringle domains of plasminogen
α-PrP	=	PrP in an α-helical conformation
β-PrP	=	PrP in an β-sheet conformation
PMCA	=	protein misfolding cyclic amplification

Figures and Tables

Figure 1 The role of plasminogen in PrP^Sc propagation. The effect of plasminogen (Plg) was assessed by PMCA using normal brain material supplemented with or without 0.5 µM human Glu-Plg (A–D) or using Plg-deficient (Plg^-/-) brain material (F and G). Pre- (−) and post- (+) PMCA samples were treated with proteinase K (PK) and analyzed by western blotting. Seeds for PMCA were diluted either as indicated or 1:900 (G) −8,100 (F). (A) Stimulation of PrP^Sc propagation by Plg. (B) Plg-supplemented PMCA in the absence of SBH seeds. (C) Plg-supplemented PMCA in the absence of NBH. (D) Comparison of PrP^Sc levels in Plg-supplemented PMCA samples (during) vs. PMCA samples only incubated with Plg prior to PK digestion (after). (E) Comparison of PrP^Sc levels of ScN2a cell lysate after incubation with or without Plg prior to PK digestion. (F) PMCA with brain material of Plg^-/- mice and genetically unaltered littermate controls (C). (G) Restoration of PMCA using Plg^-/- brain material with Plg-supplementation. NBH, normal brain homogenate; SBH, sick brain homogenate; PrPKOBH, brain homogenate of PrP^C-deficient mice; CL, cell lysate. Reproduced with permission from The FASEB Journal, Mays and Ryou 2010.35

Figure 2 PrP^Sc propagation increased by plasminogen in prion-infected cells. (A) The levels of PrP in ScN2a cells incubated with 0–0.5 µM human Glu-plasminogen (Plg) for two days. (B) The levels of PrP in ScN2a cells incubated with 0, 0.1 and 1.0 µM Plg or the first three kringle domains of Plg [K(1+2+3)] for six days. (C) The levels of 3F4-tagged PrP^C and nascent PrP^Sc formation in ScN2a cells transiently transfected (Tfx) with plasmids encoding the 3F4-tagged PrP gene (PrP-3F4) and with an empty vector (mock). The transfected cells were treated with 0 or 1 µM K (1+2+3) for three days. (D) The levels of PrP in Elk21⁺ cells incubated with 0 and 0.5 µM Plg for two days. PrP was detected by anti-PrP antibody D13 (A and B), 3F4 (C) or 6H4 (D) before (−) and after (+) PK treatment. Reproduced by permission of the The FASEB Journal, Mays and Ryou 2010.35

Figure 3 Plausible mechanisms for plasminogen to enhance PrP^Sc propagation. Plasminogen may stimulate PrP^Sc propagation via conformational alteration of PrP^C to PrP* (i), enhancement of PrP^Sc aggregation (ii), stabilization of pre-exisiting PrP^Sc aggregates (iii) or scaffolding to gather PrP^C and PrP^Sc together (iv).

Table 1 Properties of plasminogen as an auxiliary factor for PrP^Sc propagation

	Protein X	Plasminogen
Composition	Protein; macromolecules	Protein
Expression	Brain; neuron-specific	Brain; neuroblastoma cell line, expressed more in the non-CNS
Subcellular localization	Plasma membrane; lipid rafts	Extracellular matrix; lipid rafts
Association with disease		Increased protein levels in the sera of human patient with CJD
Interaction	Only with PrP^C	PrP^Sc, α-PrP, β-PrP
Binding sites on PrP	Q167, Q171, V214 and Q218 in the β2-α2 loop (164–174) and C-terminus (215–223) Dominant negative mutations on the protein X binding sites such as Q167R and Q218K inhibited PrP^Sc formation in the cultured cells and prion transmission in transgenic mice K23, K24 and K27 in the N-terminus: deletion of the N-terminal lysine cluster reduced dominant negative inhibition of PrP^Sc formation	Binds to N-terminally truncated PrP (89–230) Increased binding activity to full-length PrP (23–230) Increased binding activity to PrP with Q218K dominant negative mutation The second kringle domain of plasminogen binds to the β2-α2 loop in silico Binds to both lysine clusters located to the N-terminus (23–27) and middle (100–109) of PrP
Species specificity	Homotypic interaction with PrP^C Mouse protein X has lower binding affinity to human PrP^C in the studies with transgenic mice	Unknown Human plasminogen binds to human, mouse, bovine and ovine PrP Human and bovine plasminogen converts mouse PrP^C to PrP^Sc in PMCA
Function	An auxiliary role in conversion of PrP^C to PrP^Sc	Enhances PrP^Sc propagation facilitated by PrP conversion in PMCA
Action mechanism	Binds to PrP^C and alters PrP^C into PrP* that interacts with PrP^Sc for conversion	Unknown

CNS, central nervous system; CJD, Creutzfeldt-Jakob disease; α-PrP, PrP in an α-helical conformation; β-PrP, PrP in an β-sheet conformation; PMCA, protein misfolding cyclic amplification.