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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01324

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Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor

Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland

View larger version (30K): [in a new window]   Fig. 1. Identification of N- and C-terminal fragments of human NgR expressed in SH-SY5Y cells. (A) SH-SY5Y cells expressing human NgR were incubated with OptiMEM for the indicated times and NgR in the lysate and medium detected by western blotting with the anti-V5 antibody. (B) Following SDS-PAGE, gel slices containing either the 64 (upper) or 48 (lower) kDa band of NgR in the cell lysate were excised and incubated with (+) or without (–) PNGase F followed by western blotting with the anti-V5 antibody. In parallel, medium from a 3 hour incubation with SH-SY5Y cells expressing NgR was treated with PNGase F and NgR was detected as above. (C) Cell lysate was incubated for 3 hours in the absence (–) or presence (+) of PI-PLC and treated with PNGase F followed by western blot analysis with the anti-V5 antibody. (D) Schematic of the human NgR construct used in the present study showing the N-terminal V5 tag (red) followed by the ligand-binding domain comprising the LRRNT (green), LRR (blue) and LRRCT (grey) sub-domains. Amino acid positions (italics) correspond to those for non-tagged human NgR. The epitope of the human NgR-specific monoclonal antibody (-hNgR-328) and the C-terminal GPI-anchor (GPI) are indicated. (E) Cell lysate and medium from a 3 hour incubation with SH-SY5Y cells expressing NgR and conditioned medium from a 72 hour incubation with HEK-293T cells expressing NgR-310 were subjected to western blot analysis with either the anti-V5 antibody, human NgR-specific monoclonal antibody (-hNgR-32B) or human NgR-specific polyclonal antibody (-hNgR poly).

View larger version (33K): [in a new window]   Fig. 2. CTF-NgR is at the cell surface and associated with lipid rafts. (A) SH-SY5Y cells expressing NgR were surface biotinylated and biotinylated proteins precipitated from the cell lysate with streptavidin-agarose. NgR and clathrin were detected in the input lysate (LYS) and streptavidin-agarose precipitate (STREP PPT) using the human NgR-specific polyclonal antibody and anti-clathrin antibody, respectively. (B) Cells were homogenised in the presence of 1% Triton-100 and subjected to buoyant sucrose density centrifugation to isolate lipid rafts. Following centrifugation, the gradient was separated into eight fractions, with fraction 1 containing the highest density of sucrose. Equivalent volumes from each fraction were subjected to SDS-PAGE and western blot analysis with the indicated antibodies. NgR was detected by either the anti-V5 antibody or the human NgR-specific polyclonal antibody.

View larger version (17K): [in a new window]   Fig. 3. NTF-NgR generation is inhibited by BFA and occurs at the cell surface. (A) SH-SY5Y cells expressing NgR were incubated for 6 hours in OptiMEM in the absence (–) or presence (+) of 5 µM BFA. NgR was detected in the cell lysate and medium using the anti-V5 antibody. (B) Cells were surface biotinylated at 4°C and then incubated at 37°C in OptiMEM for the indicated times. Biotinylated proteins in the lysate and medium were precipitated with streptavidin-agarose and NgR detected with the anti-V5 antibody.

View larger version (35K): [in a new window]   Fig. 4. Elucidation of the C-terminus of NTF-NgR by mass spectrometry. (A) SH-SY5Y cells expressing NgR were incubated for 48 hours in OptiMEM. NTF-NgR was immunoprecipitated from medium using the anti-V5 antibody, subjected to SDS-PAGE and visualised by staining with Coomassie Blue. The band corresponding to NTF-NgR (*) was excised from the gel along with a control blank gel slice, digested in-gel with Lys-C and the resulting fragments analysed by MALDI-TOF mass spectrometry. Bands corresponding to the heavy (H) and light (L) chains of the anti-V5 antibody in the immunoprecipitate are indicated. (B) Table showing the predicted mass and sequence of peptides generated by Lys-C digestion of human NgR and their detection by MALDI-TOF mass spectrometry. Amino acid residues correspond to those for un-tagged human NgR. The mass of the peptide corresponding to residues 423-447 could not be predicted due to the presence of the GPI anchor. The peptide of mass 26980.21 was too large to be extracted from the gel. (C) Detail of the MALDI-TOF mass spectrometry spectra showing the peak of mass 1396.72 present in the NTF-NgR digest (lower spectrum) but not the control sample (upper spectrum) predicted to correspond to the indicated sequence. (D) Table showing the mass and predicted sequence of subunits detected following MS/MS analysis of peak 1396.72. Amino acid residues correspond to those for un-tagged human NgR.

View larger version (32K): [in a new window]   Fig. 5. Effect of protease inhibitors and MßCD on NTF-NgR release. (A) SH-SY5Y cells expressing NgR were incubated for 3 hours with OptiMEM in the absence (–) or presence of either 10 µM E-64, 10 µM AEBSF, 10 µM Pepstatin, 10 µM PKF226-967 or 10 mM NH4Cl. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody. (B) Structure of PKF226-967 and table showing IC50 values of this compound for the indicated zinc metalloproteinase family members and the shedding of TNF- from human peripheral blood mononuclear (hPBMC) cells (Kottirsch et al., 2002). (C) Cells were incubated for 3 hours with Opti-MEM in the absence (–) or presence of 10 µg/ml of the indicated recombinant human TIMP. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody. (D) Cells were incubated for 30 minutes with OptiMEM in the absence (–) or presence (+) of 10 mM MßCD with or without 10 µM PKF226-967. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody.

View larger version (18K): [in a new window]   Fig. 6. NTF-NgR binds Nogo-66 but not p75NTR and inhibits Nogo-66 binding to cells expressing NgR. (A) Medium from a 3 hour incubation with equivalent amounts of mock transfected SH-SY5Y or SH-SY5Y cells expressing NgR was subjected to immunoprecipitation with the anti-V5 antibody and equivalent amounts of precipitate incubated at room temperature for 2 hours in M-PER containing 10 nM AP (white bar) or AP-Nogo-66 (black bars). Following four washes in TNS buffer, the amount of AP activity associated with the precipitate was quantified using 1-StepTM PNPP. The results are the mean±s.d. of three independent experiments. (B) 1 nM AP or AP-Nogo-66 were pre-incubated with medium from a 3 hour incubation with mock transfected SH-SY5Y (black bars) or SH-SY5Y expressing NgR (white bars) and subsequently incubated with CHO cells stably expressing human NgR. Cells were washed, fixed and the amount of AP activity associated with the cells quantified as above. The results are the mean±s.d. of three independent experiments. (C) Lysate from SH-SY5Y cells expressing Ng R was subjected to immunoprecipitation (IP) with either the anti-p75NTR antibody or an unrelated IgG antibody. NgR and p75NTR were detected in the input lysate and immunoprecipitate using the anti-V5 and anti-p75NTR antibodies, respectively.

View larger version (55K): [in a new window]   Fig. 7. Detection of fragments analogous to NTF- and CTF-NgR in human brain cortex and CSF. NgR was detected in human brain cortex homogenate (cortex), human CSF, and cell lysate and medium from a 1 hour incubation with SH-SY5Y cells expressing NgR (hNgR:SH) by western blotting with the human NgR-specific polyclonal antibody.

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