Supplemental Information

Transcription

1 Supplemental Information Figure S. Regions recognized by the specific antibodies. The amino acid sequence of the major p3-alc species, p3-alc α 35, p3-alc β 37 and p3-alc γ 3, are indicated as red letters. Regions recognized by specific antibodies were indicated with double underline. Broken lines show the putative transmembrane (TM) region suggested by Swiss-Prot protein knowledgebase. Numbers on amino acids indicate their positions. Figure S. Quantitative accuracy of immunoprecipitation-mass spectrometric analysis. (A) Quantitative accuracy of the ratio of p3-alc α 39/p3-Alc α 35. To the fixed amount ( ng) of synthetic p3-alc α 35 peptide, various amount (5,,, 4 ng) of synthetic p3- Alc α 39 peptide was added. From the mixture in PBS containing. % (w/v) bovine serum albumin, both peptides were recovered by immnoprecipitation with UT35 and analyzed with MALDI-TOF/MS (left). The area ratios of p3-alc α 39/p3-Alc α 35 were compared to the theoretical ratio (the amount ratio of p3-alc α 39/p3-Alc α 35 used in this analysis) (right). (B) Quantitative accuracy of the ratio of p3-alc β 4/p3-Alc β 37. To the fixed amount (5 ng) of synthetic p3-alc β 4, various amount (.5, 5, 5, ng) of synthetic p3-alc β 37 peptide was added. From the mixture in PBS containing. % (w/v) bovine serum albumin, both peptides were recovered by immnoprecipitation with UT43 and analyzed with MALDI-TOF/MS (left). The area ratios of p3-alc β 4/p3-Alc β 37 were compared to the theoretical ratio (the amount ratio of p3-alc β 4/p3-Alc β 37 used in this analysis) (right). y=inclination and R =correction coefficient (n=3). Figure S3. Evidence that Alc family proteins and APP undergo identical processing by ADAM 7 α-secretase. (A-D) Generation of salc and sapp in cells expressing ADAM 7. Na cells were transiently transfected with the indicated amounts of pcdna3-adam7- HA in the presence of the indicated amounts of pcdna3-flag-halc α (panel A), pcdna3.-flag-halc β (panel B), pcdna3.-flag-halc γ (panel C), or pcdna3- FLAG-APP 695 (panel D). Empty vector was also added to standardize the amount of plasmid used for each dish. Conditioned culture medium (Medium, ml) was immunoprecipitated with the anti-flag M antibody and analyzed by Western blotting with the same antibody. Cell lysates (Cell, µg protein) were analyzed by Western blotting with M for Alc family proteins and APP, or with the anti-ha antibody for ADAM 7 (left panels). The levels of salc and sapp in cells exogenously expressing ADAM 7-HA are indicated as ratios relative to the levels of expression in cells without exogenous ADAM 7-HA expression; this was assigned a reference value of. (values represent means ± s. e). The data were analyzed by a one-way analysis of variance followed by the Tukey-Kramer multiple comparison test (n=4 for A and B, n=3 for C and D, *, p<.5; **, p<.; ***, p<.5) (right panels). imadam 7, immature ADAM 7; madam 7, mature ADAM 7.

2 Figure S4. Cleavage of APP and Alc family proteins in cell expressing BACE. HEK93 cells were transiently expressed with FLAG-APP695 (A) or FLAG-hAlcα, FLAG-hAlcβ, and FLAG-hAlcγ (B) in the presence (+) or the absence (-) of human BACE expression. Cultured medium ( ml, Medium) was immunoprecipitated with anti- FLAG M antibody and analyzed by Western blotting with same antibody (Medium in A and B). The cell lysates (cell, µg protein) were analyzed by Western blotting with anti-app/c antibody to detect APP (second row in A. mapp, mature APP; imapp, immature APP) and CTFα/β (third row in A), and anti-alcα/γ UT-83 and anti-alcβ UT- 99 antibodies to detect Alc (second rows in B) and Alc-CTF (third rows in B), or anti- BACE Ab- antibody (Oncogene product, fourth rows in A and B). Figure S5. Identification and characterization of p3-alc α, p3-alc β and p3-alc γ species secreted from cells. (A-C) Determination of the molecular mass (left panels) and amino acid sequence (middle and right panels) of p3-alc α (A), p3-alc β (B) and p3-alc γ (C). HEK93 cells ( x 6 ) were transiently transfected with 6. µg of pcdna3-halc α, pcdna3-halc β, or pcdna3-halc γ. p3-alc α (panel A), p3-alc β (panel B), and p3-alc γ (panel C) were recovered from the culture medium ( ml) by immunoprecipitation with their respective antibodies. The immunoprecipitates were analyzed by MALDI-TOF/MS to determine the molecular mass (left panels). Arrows in panel A indicate p3-alc α 35 and p3-alc α N+35. Arrows in panel B indicate p3-alc β 37 and p3-alc β 4. Arrows in panel C indicate p3-alc γ 3 and p3-alc γ 34. The signals indicated with arrows in left panels were further analyzed by MALDI-MS/MS analysis to determine the amino acid sequence (middle and right panels). The signal of p3-alc γ 34 shown in panel C was faint to determine the sequence, therefore, we used HEK93 cells expressing FAD-linked PS mutant (L66P) and performed MALDI-MS/MS analysis for p3-alc γ 34 which was also shown in right panel along with p3-alc γ 3 (middle panel) of panel C. The amino acid sequences determined by this study are indicated in Figure. Figure S6. Identification and characterization of p3-alc α, p3-alc β and p3-alc γ species in human CSF. MALDI-TOF/MS analysis of p3-alc α (A), p3-alc β (B), and p3-alc γ (C) recovered from human CSF. (A) The human CSF mixture (3 µl) was subjected to immunoprecipitation with the 3B5 antibody (5 µg IgG fraction). The precipitates were analyzed for molecular mass by MALDI-TOF/MS (left panel). The arrow shows p3- Alc α 35 whose amino acid sequence was confirmed by MALDI-MS/MS analysis (right panel) (B) The human CSF mixture (3 µl) was subjected to immunoprecipitation with UT43 ( µl serum), and the precipitates were analyzed as described above. The arrow shows p3-alc β 37 whose amino acid sequence was confirmed (right panel). (C) The human CSF mixture ( ml) was subjected to immunoprecipitation with UT66 ( µl serum), and the precipitates are analyzed as described above. The arrow shows p3-alc γ 3 whose amino acid sequence has not been determined by its small amount but the molecular size

3 was completely identical of p3-alc γ 3 as shown in Supplementary Figure SC. Amino acid sequences determined in this study are identical to those shown in Figure. Figure S7. Characterization of p3-alc α recovered from human CSF. (A) MALDI-MS/MS analysis (linear mode) of β-alc α recovered from human CSF from three subjects (Subject, CDR ; Subject, CDR.5; Subject 3, CDR ). p3-alc α was immunoprecipitated as described in Figure 5. Peaks of respective p3-alc α species are standardized with that of p3-alc α 35 and compared. The peptide indicated with asterisk (*) composing of 37 amino acids was a composite signal of p3-alc α 37 and p3-alc α N+35. (B) The signal composite of p3-alc α 37 and p3-alc α N+35 are separated respective components (left panel) with a reflector mode of MALDI-MS/MS analysis. Amino acid sequence of p3-alc α 37 and p3- Alc α N+35 are shown along with that of p3-alc α 35 (right). Figure S8. Comparison of p3-alcα species in CSF and brain. The p3-alcα species in mouse CSF (upper panel), mouse brain (middle panel) and human CSF (lower panel). Adult mice CSF ( µl) was collected from the cisterna magna by the methods of lie and Duff (J. Vis. Exp. [8]. pii;96) and subject to immunoprecipitation with UT35 in the presence of protease inhibitor cocktail. Mouse brain was homogenized in TBS (5 mm Tris-HCl [ph 7.6] and 5 mm NaCl) including protease inhibitor cocktail and subject to ultracentrifugation (, x g for 3 min) at 4 o C. The pellet was lyzed in TBS containing % (w/v) SDS and 4 M urea. The lysate was diluted by -folds volume with TBS and subject to immunoprecipitation with UT35. Human CSF (3 µl) of a subject (CDR.5) was subject to immunoprecipitation with UT35 in the presence of protease inhibitor cocktail. The immunoprecipitates were analyzed by MALDI-TOF/MS, and p3- Alc α species were identified; 34, p3-alc α 34; 35, p3-alc α 35; 36, p3-alc α 36; 37*, a mixture of p3-alc α 37 and p3-alc α N+35 (see Supplementaly Fig. S7). Amino acid sequences of p3-alc α major and minor species are completely identical between mouse and human.

4 p3-alc peptides Mass observed (Da) Mass expected (Da) Cell media p3-alca p3-alcb p3-alcg N CSF p3-alca p3-alcb Supplementary Table Molecular masses observed by TOF/MS analysis and those of expected.

5 Alca p3-alca M-A-A-Q-P-Q-F-V-H-P-E-H-R-S-F-V-D-L-S-G-H-N-L-A-N-P-H-P-F-A-V-V-P-S-T-A-T-V-V-I-V-V-C-V-S-F- TM Alcb 3B5 UT35 p3-alcb S-H-V-L-S-S-Q-Q-F-L-H-R-G-H-Q-P-P-P-E-M-A-G-H-S-L-A-S-S-H-R-N-S-M-I-P-S-A-A-T-L-I-I-V-V-C-V-G- Alcg UT43 p3-alcg N-H-L-I-V-Q-P-P-F-L-Q-S-V-H-H-P-E-S-R-S-S-I-Q-H-S-S-V-V-P-S-I-A-T-V-V-I-I-I-S-V-C-M-L-V-F-V-V-A-M- UT66 Supplementary Figure S

6 Area ratio (p3-alcb 4/37) Area ratio (p3-alca 39 / 35) A x 5.5 p3-alca y =.4565x R² = p3-alca Ratio (p3-alca 39 / 35) m/z m/z Bx 4 p3-alcb 37.5 y =.995x -.3 R² = p3-alcb m/z m/z Ratio (p3-alcb 4 / 37) Supplementary Figure S

7 sapp (Relative ratio) salcg (Relative ratio) salcb (Relative ratio) salca (Relative ratio) A FLAG-Alca (.5 g DNA) ADAM7-HA.5.5 ( g DNA) Medium salca Alca Cell imadam7 madam7 B FLAG-Alcb (.5 g DNA) ADAM7-HA.5.5 ( g DNA) 3 ** *.5.5 ADAM7-HA ( g DNA) 3 * Medium salcb C Cell ADAM7-HA Medium FLAG-Alcg (. g DNA).5.5 Alcb imadam7 madam7 ( g DNA) salcg ADAM7-HA ( g DNA) * D Cell FLAG-APP (.5 g DNA) ADAM7-HA.5.5 Medium Cell Supplementary Figure S3 Alcg imadam7 madam7 ( g DNA) sapp mapp imapp imadam7 madam ADAM7-HA ( g DNA) *** * **.5.5 ADAM7-HA ( g DNA)

8 A FLAG-APP BACE Medium sappa sappb mapp imapp Cell CTFb CTFa BACE B FLAG- Alca Alcb Alcg BACE Medium salc Alc Cell CTF BACE Supplementary Figure S4

9 A p3-alca N m/z LIFT AQPQFVHPEHRSFVDLSGHNLANPHPFAVVPSTAT m/z LIFT MAAQPQFVHPEHRSFVDLSGHNLANPHPFAVVPSTAT [ Abs. I nt. * ] [ Abs. I nt. * ] a F V H S F V H N L A N V T A b F V H S F V H N L A P E H R Db 5L P H P y V P H D H P S R F a M Q P Q F V H H F V G H N L A N b Q F V H H F V S G H N N A P E R S Db 7L y T S A H P L S H P V V R H Q y y 6 y 6 y 5 y y 3 y 5 b 5 a 34 y 6 y y a y 4 y 8 a 5 y 3 y 33 b 4 y b 9 b y 3 b 4 a 6 b 33 a y 35 b 7 b 9 a 4 a 9 a b 3 y 8 a 9 a 4 b 5 a 3 b b 34 b 3 a 7 a 9 b 8 b a 3 b 8 a 5 b 3 a b 8 a 3 a 8 a a 7 a 33 b 6 y 4 a 3 b b b 6 b 4 b y 4 y 6. 5 y 4 y y y 3 y 5 y 7. y 3 y 5 y 8 y b b 5 b 6 b 3 a 5 y 7 y a b. 5 b 8 a 5 y 9 b 5 a 3 b 4 y 6 b b y 9 a 8 a a 3 y 8 b 4 a 6. y a 4 b 6 b 9 a b 9 y 3 a b 7 b 4 b 7 a 4 b a 6. 5 a 7 a 9 b y 3 a 9 b a 4 a 7 b 3 a 5 a a a 9 b m/z B p3-alcb m/z LIFT VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT [ Abs. I nt. * ] a V L S S Q L H R G H Q P P P E G H H R P b a V L S S Q L H R G H Q P P H H Q F M S S S S M S A y T A A S P I N R H S S A L S H M P P H G F Q S S G A Q L Q L V m/z LIFT VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAATLII [ Abs. I nt. * ] a Q Q F L H Q M A S L A H M b Q F L H Q 6 S R G H P E H N I a y A R H S S A L S H GA M P P Q H G R H y y 4 4. a 3 b 3. 5 y 3 y 5 y 4 3. b 3 y 3 y 9 a 3 b 3 b 7 y 7 y. 5 b b a 7 y 3 y 6 a a 4 y b 6 a. a 3 y y 9 y y 7 y b a 6 y 35 y 5 b b y 8 y y 6 y 8 b 5 y 9 y 33 b y 4 y 6 b 8 a 8 b 7 y a 6 a a 4 b y 3 a 33 b 5 y 5 b 7 a 7 b 9 b 5. a 5 a a y 3 y 3 y 34 b 4 a 7 b 6 a 9 a 5 b a 9 b 9 b 3 b 33 y 36 b 4 b 8 b 4 b 3 a a 3 b a y 4 y 9. 6 b 3 y 7. 4 a 3 y 3 y 3 b 9. y 6 y 9 a b 8 b 6 y 8 y a. y 3 y 7 a 9 a 4 a 6 b 8 b b b 7. 8 y 3 y 5 y 6 b 7 y 3 b 3 b 5 b 9 a y 6 a 7 a 7 y y 5 y y 8 y 3 b 3. 6 b a 5 a 9 y b 6 a 4 a 7 a 3 a 8 a 8 a 36 b y 5 a 3 a 3. 4 b y 34 b b 5. b 39 y 4 b C p3-alcg 3 x m/z m/z LIFT LIVQPPFLQSVHHPESRSSIQHSSVVPSIAT m/z LIFT LIVQPPFLQSVHHPESRSSIQHSSVVPSIATVVI [ Abs. I nt. * ] [ Abs. I nt. * ] a P H S S I Q H S V b P H S H V Q S V H P E S R y Q I S S H Q H H V S L P Q y 3 a L I P P F P S I Q H S S V V b V Q P P F S H S S I Q H S V y 33I b 7 y V T A I S P S S E P H H V L V V S R S Q P b b 4 y 6 a y 9 y b a 7 y 8 5. b 9 a 7 6 y 9 5 y 5 b 8 y 7 b 4 y 7 a 8 y y 6 y 3 4 a 4 b 7 b 7 b 5 y y 3 y b 6 y b b 3 b 3 y y a 6 a b 3 a a 3 y 9 a a 9 a b 3 b b 9 y 5 b a 5 a 9 a y 4 b 6 a y 8 b a 3 a 5 b 5 b 8 b 4. 5 y 3 4. y 7 y 4 y y 6 y b 4 y 3 b a 6 y 4 y 6 3. y 3 y 9 a y 5 b 6 b 3 b 6 b 3 y 8. 5 b 5 a 3 a 6 b 7 y y 8 y a 4 b b 5. b a 7 y b 3 b 8 y 7 a a 5 y. 5 a a 8 b a 4 a 4 b 9 y 9. a 5 a a 3 b 5 a 9 a b. 5 a 8 y 6 y 34 b m/z Supplementary Figure S5

10 A p3-alca RP\5 LP6% shots\sref Raw m/z LIFT AQPQFVHPEHRSFVDLSGHNLANPHPFAVVPSTAT 4 [ Abs. I nt. * ] a V H P E H R S F V S G H N L A P F A V V T A T b V H P E H R S F V S G H N L A V V T A T A Q P Q F Db 5L y T A T V V A F P H P N A L N H G S L D S V F A R H H y 35 9 b 35 y a 35 8 y 5 y 7 y 9 y 4 7 y 6 y 8 y 3 y 6 y 8 y y 6 y 9 6 b 6 y b 4 b 8 y 3 y b y 5 y 8 y 34 5 y y 5 a 6 y 9 a 4 a 8 a y 3 y b b 5 b 34 4 b a 5 b 8 b 3 y 7 b a 9 a a 5 a 34 3 y y 3 b 4 b 7 a 3 b 7 a y 4 b 8 y 3 b 33 a b 5 a 7 a a 7 b 9 b a 8 b 3 b b 3 a 33 a 9 b b 6 a 9 b a 7 a 3 b 3 a a 8 b 9 a 6 a a 6 b 9 a m/z B p3-alcb 37 44shots\_E8\\SRef Raw m/z LIFT VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT [ Abs. I nt. * ] a V L S S H R G H Q P P P E M A G H S L S H R N b a V L S S H R G H Q P P P E M A G H S L S H R N Q Q F L S M y T A A S P I M S N R H S S A L S H G A M E P P P Q G R H Q 9. H S L V y b 3 6. a y y 4 y 4 5. y y 4 b y 3 y b y y a 4 y 5 y 8 y y 7 y 9 a 4. b b y 7 y y 6 y 8 a 3. 5 a a b 7 a 3 b 6 y 6 y 6 a y 37 a 7 3. y a 6 a 9 b 6 b 3 b 9 y 3 y 9 a 6 y 5 b 8 a. 5 y 5 a 6 a 3 a 9 y 3 y 3 y 36 y 8 y b 5 b 5 a 8. b 5 b b 8 b y 7 b 8 a 4 b 6 a 5 b 7 a 3 y b 4 a a 8 b 7 a 5 a 7 b 3 a 35 a 9 b. b 4 b b 3 b 9 y 34 b 3 a 8 b 9 b m/z m/z C p3-alcg 3 CSF\RP-HPC this\sref Raw m/z Supplementary Figure S6

11 A p3-alca 35 Subject Subject Subject 3 37* m/z B Supplementary Figure S7

12 3 5 p3-alca 35 75Mold IP mouse CSF shots\_e3\\slin 5 37* LP6 TBSppt 75Mold SDSUtrea IP mouse 75IP CSF shots\_b4\\slin shots\_e3\\slin * x LP65% K\7 65% 4 k 3shots\SLin m/z m/z 35 p3-alca * m/z Supplementary Figure S8