Dol-PP Asn Asn Asn Asn ER
Glc I&II αMan I GnT I
Golgi
KIF
- - + +
Medium
Aβ C99
Notch
KIF
--
-+ +
+
15
2
Notch
Myc-Notch-β mNCT imNCT KIF
- - + +
150250 100
50 37
(A) (B)
(C)
β-actin
sAPPβ
sAPPα
10 C83
250 100 150
mAPP
imAPP KIF
- - + +
100
PS1 CTF 100
PEN-2
20 15 10
75 50
mBACE imBACE KIF
- - + +
(D)
(E)
Fig. 13: Kifunensine reduced Aβ generation in wild type APP expressed HEK (K269) cells. (A) Kifunensine interrupt NCT maturation; (B-C) The level of Aβ reduced while Notch-β generation remained unchanged upon Kifunensine treatment; (D-E) Kifunensine reduced sAPPβ generation with no change in the level of APP, sAPPa, C83, and γ-secretase components PS1 CTF and PEN2.
KIF
- + +
C99
- - +
- + +
- - +
Cell lysate Medium
Aβ 15 C99
KIF
- + +
Notch
- - +
Myc-Notch-β Myc-Notch 15
2 (A)
(C)
(B)
50
37 β-actin
mNCT imNCT KIF
- - +
250150 100
0 50 100 150
(-) Kif (+) Kif
% of (-) Kif
Aβ/C99
N.S.
n = 4, paired t-test, N.S.; not significant (D)
Fig. 14: Kifunensine treatment did not inhibit Aβ and Myc-Notch-β production in C99 expressing HEK 293 cells. (A) Kifunensine inhibit NCT maturation. (B) Myc-Notch-β generation was not affected by Kifunensine treatment. (C-D) level of AMyc-Notch-β in the conditioned medium of Kifunensine treated HEK293 cells was also remain unaffected.
NCT PS1-CTF
PEN-2
(A)
(D)
C99-FLAG
AICD-FLAG
Notch-FLAG NICD-FLAG
C99-FLAG
Aβ 150
75
15 10
15
5 15 10 15
7 kDa
WT
(E)
Lec-1 Lec-2
(C)
WT Lec-1 Lec-2
WT Lec-1 Lec-2
0 20 40 60 80 100 120 140
WT Lec-1 Lec-2
% of WT
AICD NICD
***
P = 0.05
***
* NS P = 0.06 (B)
0 20 40 60 80 100 120
WT Lec-1 Lec-2
% of WT **
*
Fig. 15. Mutants exhibit reduced γ-secretase activity in soluble state. CHAPSO-solubilized γ-secretase of WT, Lec-1 and Lec-2 were incubated with 240 nM of substrates (C99-FLAG/Notch-FLAG) for 4 h at 37°C. After incubation, reaction mixtures were subjected to western blotting. (A) Mutant Lec-1 and Lec-2 reduced AICD with subtle effect on NICD generation compared with WT. AICD-FLAG and NICD-FLAG were detected by anti-FLAG antibody. (B) The band intensities of AICD and NICD were measured as in Fig. 11B. Lec-1 preferentially reduced C99 processing compared to Notch. (C) Similar intensities of NCT, PS1-CTF, and PEN-2 were detected in the WT and mutants. (D-E) Mutant Lec-1 and Lec-2 significantly reduced Aβ level compared to WT. The band intensity of Aβ was measured as in Fig. 11B. Data represent means ± SD (ICDs, n = 4; Aβ, n = 3; *p < 0.05; **p < 0.01; ***p <
0.001; NS, not significant; ANOVA with Scheffe’s post hoc test compared with WT). The p values on graph paired t-test. Arrowheads indicate carboxyl-terminally truncated, C99-FLAG fragments [37].
150
100
100
20 15 20 15 20 15
10 10
10
20
20
20
75 50 75 50 75 50
NCT
PS1-CTF
PEN-2
Aph-1
Flotillin WT
Lec-1 Lec-2
WT Lec-1 Lec-2
WT Lec-1 Lec-2
WT Lec-1 Lec-2
WT Lec-1 Lec-2
Input 1 2 3 4 5 6 7 8 9 10 11 12 ppt
Raft fractions
kDa
Fig. 16. No alteration of the subcellular distribution of γ-secretase subunits in mutants.
Lipid rafts were prepared from WT and mutant CHO Lec cells by sucrose density gradient centrifugation in the presence of 1% CHAPSO. γ-Secretase components were enriched in lipid raft fractions, as well as flotillin raft marker (#3 - #5). Dashed box indicates raft fractions.
WT Lec-1 Lec-2 1,236
1,048 720 480
1,236 1,048 720
PS1-CTF 480 NCT
WT Lec-1 Lec-2
(A) (B)
kDa kDa
Fig. 17. Glycosylation of NCT is not required for γ-secretase complex assembly. CHAPSO-solubilized γ-secretase of WT, Lec-1 and Lec-2 were subjected to 3-12% blue native gel analysis followed by Western blotting. 36 µg of protein was loaded to each lane. Levels of (A) PS1-CTF and (B) NCT in mutants were higher or equivalent to that of WT. NCT and PS1-CTF were detected with anti-nicastrin and anti-PS1-CTF antibodies, respectively.
250 150 100
20 15
10
mNCT
imNCT
PEN-2 PS1-CTF
WT Lec-1 Lec-2 WT Lec-1 Lec-2
Lysate IP
Fig. 18: Immunoprecipitation of γ-secretase complex. CHAPSO-solubilized membrane fractions of WT, Lec-1 and Lec-2 were immunoprecipitated with anti-PEN2 antibody. The immunoprecipitated complexes were detetcted by Western blot analysis with anti-NCT antibody for NCT, anti-PS1 CTF for PS1 CTF, anti-PEN2 for PEN2 respectively. Lysate and IP indicate solubilized membrane fractions and immunoprecipitated complexes, respectively.
PS1-CTF
PEN-2
Aph-1
C99-FLAG imNCT kDa mNCT
150 75 20
15
20 10
15
1/1 1/2 1/4 1/8 1/16 Mock WT Lec-1 Lec-2 Input
0 0.5 1 1.5
WT Lec-1 Lec-2
Intensity (AU)
PS1 CTF
N.S.
Scheffe post hoc test compared with WT; N.S.; not significant; n = 3
(A)
(B)
Fig. 19. Glycosylation deficiency of NCT induced γ-secretase cleavage deficiency but not substrate recognition. Purified C99-FLAG that was recaptured with M2 anti-FLAG agarose beads was incubated with CHAPSO-solubilized γ-secretase of WT, Lec-1 and Lec-2 at 4°C overnight. After thorough washing, the beads were subjected to Western blotting to visualize γ-secretase components. The intensities of the coimmunoprecipitated γ-secretase components
was not affected in the mutant Lec-1 and Lec-2 samples (upper panel). The level of C99-FLAG bound to the beads did not alter in the mutants (lower panel). The data are representative results of three independent experiments. Arrowhead indicates nonspecific reaction with the antibodies. Only beads and γ-secretase was incubated without C99-FLAG in mock.
Normal glycosylation NCT Maturation
Proper conformation of NCT
Favors substrate recognition and cleavage
Interrupted glycosylation
Immature/partially matured NCT Normal assembly of γ-secretase Improper conformation of NCT ? γ-Secretase cleavage defect
Mutant cells Parental cells
(A) (B)
Fig. 20: Schematic showing overall finding of this study. (A) In parental cells, NCT achieve proper conformation through complete glycosylation which favors substrate recognition and cleavage. (B) In mutant cells, NCT are immature or partially mature because of interrupted glycosylation which may cause conformational change of NCT resulting in γ -secretase cleavage defect.
Fig. 21: Structure of tetraspanin. Tetraspanins are transmembrane glycoprotein containing four transmembrane domains (TM, green cylinder). TM1 and TM2 flank small extracellular loop (SEL), and TM3 and TM4 flank large extracellular loop (LEL). Tetraspanins contain several conserved amino acid residues like CCG motif which forms disulfide bridges with additional conserved cysteines (black line).
Adopted from (S. Levy and T. Shoham, 2005) [39].
Searched for non-raft tetraspanins by proteomics analysis
Non-raft tetraspanins were identified by proteomics analysis, and their non-raft
association was confirmed by sucrose density gradient analysis
CHAPSO-soluble membrane fractions of PS1/2 dKO MEF cells
Transfection into HEK (K269) cells and measured effect on A β generation Prepared TSPAN7 overexpressing HEK
(K269) cell line, and examined A β generation upon TSPAN7 overexpression
Fig. 22: Flowchart showing experimental procedure used in chapter-3
Tetraspanin7 (TSPAN7)
METKPVITCKTLLIIYSFVFWITGVILLAVGVWGKLTLGTYISLIAENS
TNAPYVLIGTGTTIVVFGLFGCFATCRGSPWMLKLYAMFLSLVFLAELVAGIS GFVFRHEIKDTFLRTYTDAMQNYNGNDERSRAVDHVQRSLSCCGVQNYTN WSSSPYFLDHGIPPSCCMNETDCNPLDLHNLTVAATKVNQKAVDWHAAGLL SVPVHHCQSV
Tetraspanin8 (TSPAN8)
MAGVSSCLKYSMFFFNFLFWVCGTLILGLAIWVRVSKDGKEIITSGDSSTNP FIAVNILIAVGSIIMVLGFLGCCGAVKESRCMLLLFFIGLLLILILQVAAGILGAAF KPEYNRILNETLYENAKLLSDNTDEAKDFQKAMIVFQSEFKCCGLENGAAD WGNNFVEAKESCQCTGTDCATYQGSSVYPKTCLSLIKDLFEKNIIIVIGIAFG LAVIEILGLVFSMVLYCQIGSK
CD82
MGAGCVKVTKYFLFLFNLLFFILGAVILGFGVWILADKNSFISVLQTSSSSLQ VGAYVFIGVGAITIVMGFLGCIGAVNEVRCLLGLYFVFLLLILIAQVTVGVLFYF NADKLKKEMGNTVMDIIRNYTANATSSREEAWDYVQAQVKCCGWVSHYN WTENEELMGFTKTTYPCSCEKIKEEDNQLIVKKGFCEADNSTVSENNPED WPVNTEGCMEKAQAWLQENFGILLGVCAGVAVIELLGLFLSIC
LCRYIHSEDY SKVPKY CD63
MAVEGGMKCVKFLLYVLLLAFCACAVGLIAIGVAVQVVLKQAITHETTAGSLL PVVIIAVGAFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAVAIAGYVFR DQVKSEFNKSFQQQMQNYLKDNKTATILDKLQKENNCCGASNYTDWENIP GMAKDRVPDSCCINITVGCGNDFKESTIHTQGCVETIAIWLRKNILLVAAAAL GIAFVEVLGIIFSCCLVKSIRSGYEVM
(A)
(B)
Flotillin 50
37 25 37
37 25
50 37
CD82 TSPAN7
TSPAN8
CD63
Input 1 2 3 4 5 6 7 8 9 10 11 12 ppt Raft fractions
N/S
Fig. 23: Identification of non-raft tetraspanins. (A) proteomics analysis identified peptide sequences of TSPAN8, CD82, CD63 and TSPAN7 in the CHAPSO-soluble membrane fractions of PS1/2 dKO MEF cells (highlighted in red); (Funamoto et al., unpublished data). (B) sucrose density gradient analysis of HEK (K269) cells homogenate followed by Western blotting revealed that identified tetraspanins were predominantly accumulated in the non-raft denser fractions (#7 to #12). Expected molecular weight CD82 (29.6 kDa), CD63 (43-53 kDa), TSPAN8 (26.06 kDa), and TSPAN7 (37.11 kDa) . Lipid raft fractions were determined by the presence of lipid raft marker flotillin. Raft fractions were indicated by the rectangle dashed box. ppt and N/S indicates pellets and non-specific interaction with antibody, respectively.
-
-HEK/APP (K269)
MediumCell Lysate
Aβ
C99
Intra Aβ 5
15
5
n = 4, **** p < 0.001, Paired t-test.
TSPAN7
+ +
37 TSPAN7
0 20 40 60 80 100 120
Mock TSPAN7
% of mock
****
(A)
(B)
50
37 β-actin
Fig. 24: TSPAN7 reduced Aβ level in APP expressing HEK (K269) cells. (A-B) Transfection of TSPAN7 into K269 cells and measured effect on Aβ generation. TSPAN7 significantly reduced Aβ level with no change in the level of C99. Intracellular Aβ tends to reduce as well.
pcDNA
Mock TSPAN7 Cl-20 stable. ex.
0 50 100 150
Mock pcDNA TSPAN7 Cl-20
(%) of mock
Extracellular Aβ HEK/APP (K269)
100
5 5
37
100 15
*** ***
Scheffe post-hoc test **P <0.01, ***P < 0.001;
N.S.; not significant; n = 3.
100
5 5
37
100
15
0 20 40 60 80 100 120
Mock pcDNA TSPAN7 Cl-20
% of mock
Intracellular Aβ
N.S.
APP Aβ
Intra Aβ TSPAN7
sAPPβ
C99
(A)
kDa
(B)
(C)
β-actin
50 37
Fig. 25: TSPAN7 overexpression reduced both intracellular and extracellular Aβ level.
(A) levels of APP, C99 and sAPPβ were not changed while intracellular and extracellular Aβ levels reduced upon TSPAN7 overexpression in HEK (K269) cells. Cl-20 harboring stable overexpression of TSPAN7. (B-C) both transient and stable expression of TSPAN7 reduced both intracellular and extracellular Aβ generation. The band intensity of Aβ was measured as described in chapter-2. Data represent means ± SD of three independent experiments.
Scheffe’s post hoc test; NS, not significant; ***p <0.001.
TSPAN7
Myc-Notch-β 37
2
pcDNA
Mock TSPAN7 Cl-20 Stable ex.
Myc- Notch
Myc-Notch
15 Myc-Notch 200
4060 10080 120140 160
% of Myc-Notch
Nβ/Myc-Notch
N.S.
Scheffe post-hoc test; N.S.; not significant; n = 3.
Mock TSPAN7
Cl-20 Stable ex.
NCT
PS1-CTF 10 150
100 20 15
10
100
15
PEN-2 (A)
(B)
(C)
β-actin 50
37
Fig. 26: TSPAN7 overexpression reduced Myc-Notch processing, with no change in the level of γ-secretase components. (A-B) both transient and stable expression of TSPAN7 reduced Myc-Notch-β production. (C) No change in the level of γ-secretase components was seen upon TSPAN7 overexpression.