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Transcript
Supplementary methods
1.
Purification and cloning of Aβ aggregation inhibitory activity
Streptomyces sp. KK565 was cultured at 30C in TSBY media (3% tryptic soy bean, 0.5%
yeast extract, 1.75% dextrose, and 1% soluble starch) for 3 days with shaking at 150 rpm.
The culture broth of Streptomyces sp. KK565 was extracted with 30% methanol and the
aqueous fraction was filtered through a 10-kDa cutoff filter (Vivaspin concentrator). The
retentate (>10 kDa) was concentrated (100-fold) and dialyzed against 20 mM Tris–HCl buffer,
pH 7.5 and fractionated on a Mono-QTM anion exchange column (GE Healthcare). The
proteins were eluted in a linear gradient of 0–1.0 M NaCl. The Aβ aggregation inhibitory
activity was monitored by Congo red assay. The activity was eluted in ~0.3 M NaCl fractions
that contained a 30 kDa protein as a major component identified on a 10 %-polyacrylamide
gel. The 30 kDa protein band was transferred to PVDF membrane and the N-terminal amino
acid sequence was determined (Korea Basic Science Institute, Daejeon, Korea).
The N-terminal amino acid sequence of the 30-kDa protein, –APDIPVA–, matched the
conserved N-terminal sequences of SGAP (P80561, NCBI) and aminopeptidase from S.
coelicolor (NP_626871, NCBI). Using a forward primer targeting the N-terminus (5'GCGCCCGACATACCGGTAGCC-3') and a reverse primer targeting a region conserved
among bacterial aminopeptidases, KAKLDAAG (amino acid 42–49 of SGAP, 5'GGCGGCGTCCAGCTTGGCCTT-3'),
a
115-bp product
was
amplified from
the
Streptomyces sp. KK565 genomic DNA. The remainder of the gene was cloned using an
inverse PCR method [1, 2] (SolGent Co. Ltd., Daejeon, Korea). Streptomyces sp. KK565
genomic DNA was digested with BamH1, PvuII, and KpnI, and circularized by ligation. The
unknown flanking region was amplified with primers designed from the 115-bp product
sequence:
5'-CTACAAGGCGTCCATCGACTA-3'
and
5'-
GATCGACTGGAGCTGGGTGAG-3'. A 1.2-kb BamH1 clone containing the first 74 amino
acid coding region was sequenced and subsequent primers were designed. By repeating
inverse PCR and sequencing the products, the 1405-bp DNA containing the full-length SKAP
coding region (438 amino acid) was obtained (NCBI, DQ241794).
2. Recombinant SKAP production
The partial SKAP DNA encoding amino acid 39–320, which was assumed to be a secreted
form, was cloned in pET-26b(+) vector (Novagen), which contains the pelB leader signal for
secretion and (His)6-tag for purification. Synthesis of rSKAP was induced with 1 mM
isopropyl β-D-thiogalactopyranoside for 15 h, and then the culture was centrifuged at
10,000g for 10 min. The supernatant containing secreted rSKAP was loaded onto a Ni-NTA
(nickel-nitrotriacetic acid) Agarose (Qiagen) column (1.7  5 cm) and was washed with 5
column volumes (CVs) of 50 mM Tris–HCl buffer, pH 8.0 (buffer A) followed by 3 CVs of 2
mM imidazole in buffer A. The rSKAP was eluted with 250 mM imidazole in buffer A and
dialyzed against 10 mM Tris–HCl buffer, pH 8.
3. SDS-PAGE and immunoblotting
For quantifying rSKAP the eluents from the Ni-NTA column were separated by 12.5% SDSPAGE and stained with Coomassie brilliant blue or transferred to nitrocellulose membranes
for immunoblotting. To measure the GCPII and sAPP protein levels, cells were harvested in a
lysis buffer containing 50 mM Tris (pH7.4), 150 mM NaCl, and 1% SDS or the culture media
were collected. The cell lysate or the culture media contaning β-mercaptoethanol was boiled
for 5 min and loaded on 8 % SDS-poly-acrylamide gels (for GCPII) or on 4-16 % gradient
NuPAGE gel (for sAPPs; Invitrogen). The separated proteins were transferred onto
nitrocellulose membranes (Hybond ECL, Amersham Biosciences) which were blocked with
TBS containing 0.05% Tween-20 and 5% skim milk for 60 min at room temperature. The
blots were incubated with an appropriate primary antibody [for SKAP, anti-6 His antibody
(1:1000, Santa Cruz Biotechnology) or anti-SKAP antibody (1:1000); for GCPII, anti-GCPII
antibody (1:1000, Maine Biotechnology, Portland, ME) followed by an appropriate secondary
antibody (horseradish peroxidase-conjugated IgG anti-mouse or -rabbit antibody, Pierce).
Immunoreactive bands were detected with chemilluminescence system (Amersham) and
analysed by densitometry using the program Image J (NIH, Bethesda. MD).
4. MALDI-TOF analysis of cleaved Aβ fragments
Aβ1–40 or Aβ1–42 peptides (50 μM) were incubated with rSKAP at 37C in 20 μl of 10 mM
Tris–HCl (pH 8.0) containing 1 mM CaCl2. Aliquots (1 μl) of the reaction mixture were
taken at intervals and mixed on the sample plate with 9 μl of the crystallization matrix: 500 μl
acetonitrile, 500 μl 0.1% (v/v) trifluoroacetic acid, and 10 mg of α-cyano-4-hydroxycinnamic
acid (CHCA). Then 1 μl was applied onto the matrix and dried. The Aβ cleavage was
measured by assisted laser desorption ionization time of flight (MALDI-TOF) mass
spectroscopy (Voyager-DE STR; Applied Biosystems).
5. Measurement of cell viability
To measure the cell viability 50 μl of 2 mg/ml MTT solution prepared in PBS was added to
each 24-well of the culture plate. After incubation for 2–3 h at 37C in a humidified CO2
incubator, the medium was removed and the MTT-formazan crystals were dissolved in 200 μl
of DMSO. The absorbance at 570 nm was measured using a microplate reader. For DAPI
staining, cells were fixed in 4% paraformaldehyde solution at room temperature for 30 min
and stained with 1 μg/ml DAPI 30 min. The morphology of the nuclei was observed by
fluorescence microscopy at an excitation wavelength of 350 nm.
6. Cloning of human GCPII
To clone human GCPII cDNA, total RNA was isolated from U87 MG human astrocyte cells
and reverse-transcribed as described previously [3]. The hGCPII cDNA was amplified by
PCR using forward (5’-GATGTGGAATCTCCTTCACGAAAC-3’) and reverse (5’ATCCTCTTAGGCTACTTCACTCAAAG-3’) primers and cloned into the pcDNA3 vector
(pcDNA3-hGCPII). The hGCPII clones were verified by DNA sequencing.
Supplementary Figure
**
**
Figure S1. N-terminally truncated Aβ11–40 or Aβ11–42 did not show toxic effect on rat primary
cortical neurons.
Dissociated cells from cortexes of 16-day-old embryonic rat brains were plated in 12-well
dishes (1 × 105 cells per well) coated with poly-D-lysine (0.1 mg/ml) and laminin (20 g/ml)
and maintained in neurobasal media supplemented with B27 (Invitrogen) as previously
described. The cultured neuronal cells were treated with 10 μM of Aβ peptides for 3 days and
the cell viability was measured using MTT assay in triplicates. Three separate experiments
yielded similar results.
** < 0.01
References
[1] Ochman, H., Gerber, A. S., Hartl, D. L. (1988). Genetic applications of an inverse
polymerase chain reaction.Genetics. Genetics. 120, 621-623.
[2] Triglia, T., Peterson, M. G.., Kemp, D. J. (1988). A procedure for in vitro amplification of
DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16,
81-86.
[3] Jo, C., Kim, H., Jo, I., Choi, I., Jung, S. C., Kim, J., Kim, S. S., and Jo, S. A. (2005)
Leukemia inhibitory factor blocks early differentiation of skeletal muscle cells by activating
ERK. Biochim. Biophys. Acta. 1743, 187-197