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First published online 15 May 2007
doi: 10.1242/jcs.03459

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Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins

¹ Ludwig Institute for Cancer Research, Hospital Alemão Oswaldo Cruz, São Paulo, Brazil
² Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
³ Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brazil
⁴ Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba, Brazil
⁵ Departamento de Biologia Celular, Universidade Federal do Paraná, Curitiba, Brazil
⁶ INFAR, Universidade Federal de São Paulo, São Paulo, Brazil

View larger version (27K): [in this window] [in a new window]   Fig. 1. Vn binds PrPC in vitro and other PrPC ligands can compete for this interaction. (a) Autoradiogram of overlay assay. Indicated amounts of Ln, Fn, Vn, type IV collagen (Col) and BSA were adsorbed onto a membrane and allowed to bind to 125I-His6-PrPC. (b) PrPC-coated wells were incubated with 125I-Vn at the indicated concentrations. Wells were washed and radioactivity was measured. Scatchard plot is shown as an inset. The data represent mean ± s.d. (c) Competition assay in which 125I-Vn was incubated over PrPC-coated wells in the presence of increasing concentrations of unlabeled Vn, STI1, Ln or BSA. Bound 125I-Vn differed from addition of 5 µg unlabeled Vn (*P<0.001), addition of 10 µg unlabeled Vn or Ln (**P<0.001), or addition of 25 µg unlabeled Vn, Ln or STI1 (***P<0.001) according to Tukey's Test. (d) Competition assay in which 125I-Vn was incubated over PrPC-coated wells in the presence of increasing concentrations of unlabeled STI1 peptide or Ln -1 peptide. The percentage of Vn binding was reduced in the presence of 20 or 40 µM STI1 peptide (*P<0.02, Student's t-test).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Mapping the binding sites within Vn and PrPC. (a) 125I-Vn was incubated over PrPC coated wells in the presence of PrPC peptides. Binding to PrPC was set as 100% and binding in the presence of peptides was expressed as a percentage thereof. PrPC peptides 103-122 and 113-132 interfered with binding to Vn (*P<0.01, Student's t-test). (b) 125I-Vn binding to deletion mutant proteins 105-112, 113-119, or 105-128 His6-PrPC was markedly reduced relative to binding to wild-type PrPC, which was set as 100% (*P<0.01, Student's t-test). 51-90 and 120-125 His6-PrPC proteins exhibited 125I-Vn binding that did not differ significantly from the wild-type protein. (c,d) The hydropathy plots compare the mouse PrPC amino acid sequence from a.a. 104 to 127 and human Vn peptides with complementary hydropathy pattern: (c) Vn262-275 and (d) Vn309-322. (e) 125I-Vn was incubated in PrPC-coated wells in the presence or absence of the indicated concentrations of Vn peptides and radioactivity levels were determined. 125I-Vn binding was disrupted in the presence of Vn309-322Hu and Vn307-320Mo (*P<0.01 vs binding to PrPC alone, Student's t-test) at the two higher concentrations tested. (f) Binding of peptide Vn307-320Mo to immobilized PrPC. Wells were coated with wild-type, 105-119, 113-119 or 105-128 His6-PrPC proteins and incubated with 1 µM 125I-Vn307-320Mo. 125I-Vn307-320Mo binding to immobilized PrPC was reduced in 105-128, 105-119 or 113-119 His6-PrPC proteins relative to wild-type PrPC controls (*P<0.001, Tukey's test). Inset shows an overlay assay, where His6-PrPC or PrPc peptides 43-62 and 103-122 spotted into a membrane were incubated with biotin labeled Vn307-320Mo followed by streptavidin-HRP.

View larger version (128K): [in this window] [in a new window]   Fig. 3. PrPC and Vn expression in mouse embryos. E12.5 Prnp+/+ sagittal (a-f) or coronal (g-l) sections reacted with anti-PrPC mouse serum (a,b,c,g,h,i), rabbit serum anti-Vn (d,e,f,j,k,l) or non-immune mouse or rabbit serum (insets in panels a and j, respectively). E12.5 ZrchI Prnp0/0 mouse sagittal sections reacted with anti-PrPC mouse serum (m and n) or anti-Vn serum (panel o). Br, brain; Ht, heart; Lu, lung; Sp, spinal cord; Ga, dorsal root ganglia.

View larger version (141K): [in this window] [in a new window]   Fig. 4. PrPC and Vn colocalize in embryonic DRG. Confocal microscopy images of E12.5 mouse sagittal sections reacted with anti-Vn rabbit serum (green) and anti-PrPC mouse serum (red). Superimposed red and green images are shown in the merge column. The top row of images (a) shows three ganglia in a low magnification (as indicated by the dotted lines); the middle row of images (b) shows a single ganglion in a higher magnification; and the bottom row (c) shows a growing nerve region.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Vn binds PrPC in vivo. (a) Confocal images of dissociated DRG cells treated with Alexa 568-Vn (red) and immunolabeled with anti-PrPC (green) are shown in the top row. Confocal images of SN56 cells transfected with GFP-PrPC (green) and treated with Alexa 568-Vn (red) are shown in the bottom row. (b) Pull down assay from cell extracts incubated with Vn-Sepharose. Western blots of Vn-Sepharose-bound proteins from untransfected (lanes 1 and 4), GFP transfected (lanes 2 and 5) or GFP-PrPC transfected (lanes 3 and 6) HEK293 cells. Blots immunolabeled with anti-GFP (lanes 1 to 3) or anti-PrPC (lanes 4 to 6) antibodies revealed that PrPC, but not GFP alone, binds to Vn.

View larger version (66K): [in this window] [in a new window]   Fig. 6. PrPC-Vn interaction supports axonal growth in DRG from E12.5 mouse embryos. ZrchI Prnp+/+ (a-d) and ZrchI Prnp0/0 (e-g) DRG were cultured on poly-L-lysine-coated coverslips (c), 200 nM Vn (a and g), 0.4 µM peptide Vn307-320Mo (b and f), 0.4 µM peptide Vn161-174 (d and e) and Vn plus anti-recombinant PrPC antibody 13 µg/ml or Vn plus irrelevant IgG 13 µg/ml (h). Inset dark-field image in a shows a DRG subjected to anti-PrPC immunohistochemistry. (h) Comparison of mean axonal growth per DRG from ZrchI Prnp+/+ (white bars) and Prnp0/0 mice (grey bars) for the conditions described above. *P<0.001 vs Pll control, Tukey's Test. (i) Comparison of mean axonal growth per DRG of at least 12 ganglia from Npu Prnp+/+ (striped bars) and Prnp–/– mice (black bars) for each of the following conditions: Pll, 200 nM Vn, 0.2 or 0.4 µM Vn307-320Mo, 0.4 µM Vn161-174, Vn plus irrelevant IgG or 0.6 µg/ml anti-PrPC peptide 106-126. *P<0.001 vs Pll control, Tukey's Test. (j) The percentage of cells from ZrchI Prnp+/+ (white bars) and ZrchI Prnp0/0 (grey bars) dissociated DRG neurons that grow axons increased with increasing concentrations of Vn; *P<0.001 vs Pll control, Tukey's test.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Integrins compensate for the absence of PrPC to support Vn-induced axonal growth. (a) VnRGD peptide abrogates Vn-induced axonal growth in cultured ZrchI Prnp0/0 (grey bars) DRGs at lower concentrations (8 µM) than that (12 µM) necessary for the same effect in Prnp+/+ (white bars) DRGs. Treatment with the irrelevant peptide Vn161-174 (12 µM) had no effect. *P<0.001 vs poly-L-lysine (Pll) Tukey's test. (b) Exposure to adsorbed VnRGD-BSA peptide (0.5, 5, 1 or 10 nmol) increased axonal growth in DRG cells from ZrchI Prnp0/0 mice, but not from ZrchI Prnp+/+ mice. *P<0.001 vs Pll control, Tukey's test. (c) Adsorbed VnRGD-BSA peptide (1 nmol) induced axonal growth in cultured DRG cells from Npu Prnp–/– mice (black bars) whereas no axonal growth was present at this concentration of absorbed VnRGD-BSA peptide in Npu Prnp+/+ cells (striped bars). *P<0.001 vs Pll control, Tukey's test. (d) ZrchI Prnp0/0 dissociated DRG cells exhibited greater WOW-1 immunoreactivity than those from ZrchI Prnp+/+ mice, indicating that the knockouts had greater levels of activated v3 integrin. *P<0.001 vs Prnp+/+, Mann-Whitney's test. (e) Npu Prnp–/– dissociated DRG cells exhibited greater AP5 immunoreactivity than Npu Prnp+/+, indicating that the knockouts had greater levels of activated 3 integrin. *P<0.001 vs Npu Prnp+/+, Mann-Whitney's test.

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