Anti-prion activity found in beetle grub hemolymph of Trypoxylus dichotomus septentrionalis

Abstract

No remedies for prion disease have been established, and the conversion of normal to abnormal prion protein, a key event in prion disease, is still unclear. Here we found that substances in beetle grub hemolymph, after they were browned by aging for a month or heating for hours, reduced abnormal prion protein (PrP) levels in RML prion-infected cells. Active anti-prion components in the hemolymph were resistant to protease treatment and had molecular weights larger than 100 kDa. Aminoguanidine treatment of the hemolymph abolished its anti-prion activity, suggesting that Maillard reaction products are enrolled in the activity against the RML prion. However, levels of abnormal PrP in RML prion-infected cells were not decreased by incubation with the Maillard reaction products formed by amino acids or bovine serum albumin. The anti-prion components in the hemolymph modified neither cellular or cell-surface PrP levels nor lipid raft or autophagosome levels. The anti-prion activity was not observed in cells infected with 22 L prion or Fukuoka-1 prion, suggesting the anti-prion action is prion strain-dependent. Although the active components of the hemolymph need to be further evaluated, the present findings imply that certain specific chemical structures in the hemolymph, but not chemical structures common to all Maillard reaction products, are involved in RML prion formation or turnover, without modifying normal PrP expression. The anti-prion components in the hemolymph are a new tool for elucidating strain-dependent prion biology.

Keywords: AGE, advanced glycation end product; Advanced glycation end product; Aminoguanidine; Insect hemolymph; Maillard reaction product; PrP, prion protein; PrPc, normal cellular PrP; PrPres, protease-resistant abnormal PrP; Prion; Strain dependency.

Fig. 1

Anti-prion activities of beetle grub hemolymph. (A) Immunodetection of PrPres in ScN2a cells treated with fresh (no aging) or aged hemolymph (4 °C, 1 or 2 months). Signals for β-actin are shown as controls for the integrity of the samples used for PrPres detection. Molecular size markers on the right indicate sizes in kDa. (B) Immunodetection of PrPres in ScN2a cells treated with the hemolymph that had been heated at 37 °C or 70 °C for 17 h. (C) Immunodetection of PrPres in ScN2a cells treated with the hemolymph that had been heated at 70 °C for various lengths of time. (D) Immunodetection of PrPres in ScN2a cells treated with the browned hemolymph that had been treated with proteinase K [PK (+)] or that had been left untreated [PK (−)]. Browned hemolymph was prepared by heating hemolymph at 70 °C for 17 h. (E) Immunodetection of PrPres in ScN2a cells treated with the retentate or filtrate of browned hemolymph after ultrafiltration through membranes with different molecular pore sizes. Designated amounts of filtrates or filled-up retentates with PBS to the original volumes were added into 10 mL of each cell culture medium.

Fig. 2

Characterization of anti-prion components in beetle grub hemolymph. (A) Immunodetection of N(6)-carboxymethyl lysine in hemolymph that was heated at 70 °C for 17 h in the presence (+) or absence (−) of aminoguanidine (AG). Untreated hemolymph (No-treat) is also shown. Weak immunoreactive signals in the untreated hemolymph may represent a certain amount of N(6)-carboxymethyl lysine, presumably produced during the heat inactivation treatment, which was done immediately after sample collection. The data from two independently collected hemolymph samples are shown. (B) Immunodetection of PrPres in ScN2a cells treated with the hemolymph samples shown in (A). (C) Maillard reaction products of amino acids and BSA, and immunoblot data of PrPres in ScN2a cells treated with the Maillard reaction products [Glucose (+)]. Immunoblot data are also shown for PrPres in ScN2a cells treated with the Maillard reaction products made in the presence of aminoguanidine [Glucose (+) & AG (+)]. As negative controls, immunoblot data are shown for PrPres in ScN2a cells treated with respective amino acids or BSA in the absence of glucose [Glucose (−)] or treated with glucose alone (G), glucose plus aminoguanidine (G+AG), or aminoguanidine alone (AG). Cont* indicates a non-heated sample containing both BSA and glucose. ScN2a cells were treated by adding 0.2% (v/v) of each reaction solution to the culture medium.

Fig. 3

Effects of browned hemolymph on PrPc expression and other prion strains. (A) Immunodetection of total cellular PrPc in N2a cells treated with the hemolymph that had been heated at 70 °C for 17 h, or not heated. Cells were treated by adding 0.1% (v/v) of each reaction solution to the culture medium. (B) Flow cytometry of cell surface PrPc (Anti-PrP) and lipid raft microdomains (Cholera-toxin B) in N2a cells treated with hemolymph that had been heated at 70 °C for 17 h, or not heated. The broken line peaks on the left show their respective isotype controls. Cells were treated as described in (A). (C) Immunodetection of PrPres in three distinct prion-infected cell lines treated with hemolymph that had been heated at 70 °C for 17 h, or not heated. Cells were treated as described in (A). (D) Immunodetection of autophagosome-related LC3-II in ScN2a cells treated with hemolymph that had been heated at 70 °C for 17 h, or not heated. A trehalose-treated cell sample (TRE) is shown as a positive control. Cells were treated as described in (A). (E) Immunodetection of PrPres and PrPc in the cell lysates treated with the browned hemolymph. ScN2a and N2a cell lysates were treated with the browned hemolymph at designated concentrations at 37 °C for 1 h, and subsequently ScN2a cell lysates were digested with proteinase K. Then, PrPres in ScN2a cell lysates and PrPc in N2a cell lysates were detected. Browned hemolymph was prepared as described already.