Supporting Online Material for
A Key Enzyme in the Biogenesis of Lysosomes Is a Protease That
Regulates Cholesterol Metabolism
Katrin Marschner, Katrin Kollmann, Michaela Schweizer, Thomas Braulke,* Sandra Pohl
*To whom correspondence should be addressed. E-mail: email@example.com
Published 1 July 2011, Science 333, 87 (2011)
This PDF file includes:
Materials and Methods
Figs. S1 to S5
Material and Methods
Antibodies. Polyclonal rabbit antibodies against the human β-subunit of the GlcNAc-1-
phosphotransferase were described previously (26). Monoclonal antibodies against protein
disulfide isomerase (PDI), MnSOD and GM130 were obtained from Stressgen, Biomol and BD
Biosciences, respectively. The monoclonal (clone 9B11) and polyclonal antibodies against c-myc
were purchased from Cell Signaling Technology and Sigma, respectively. Mouse cathepsin Z
antibody was purchased from R&D Systems. Secondary antibodies conjugated to horseradish
peroxidase and fluorochrome-conjugated antibodies were purchased from Dianova and
Invitrogen. The single-chain antibody fragment scFv M6P has been described previously (18).
The monoclonal antibody 6C4 against bis(monoacylglycero) phosphate (BMP) was a generous
gift by Dr. J. Gruenberg (University of Geneva, Geneva, Switzerland). Mouse monoclonal
antibodies against LAMP2 (clone UH3) were from the Developmental Studies Hybridoma Bank
(University of Iowa).
Generation of α α/β β-subunit precursor and S1P constructs. The expression vector coding
human His-tagged α/β-precursor was generated as previously described (26) and was used as
template to generate α/β-precursor miniconstructs by overlap extension PCR. In the first step,
two simultaneous PCR reactions were performed with primers 1 and 2 to amplify the region
encoding amino acids 1 to 430, and with respective primers 3 and 4 to amplify the region
encoding amino acids 820/849/889/909/919 to 1256. In the final PCR reaction equal amounts of
the two products from the first PCR were mixed with primers 1 and 4. For amino acid
substitution in the α/β-subunit precursor constructs designed mutagenic primers and Phusion®
Polymerase (Peqlab Biotechnology) were used. The myc-tagged cDNA of human MBTPS1 in the
expression vector pCMV6-Entry was purchased from OriGene Technologies. All expression
vectors were commercially sequenced (Seqlab, Göttingen). Primer information is available on
Cell culture and transfection. BHK cells, HeLa cells and mouse embryonic fibroblasts (MEF)
of wildtype (wt) and MLII mice were cultured in DMEM (Invitrogen) supplemented with 10%
fetal calf serum (FCS; PAA Laboratories) and penicillin/streptomycin (Invitrogen). Cells grown
on 6-cm plates were transfected with cDNAs of α/β-subunit precursor and S1P constructs using
jetPEI™ Transfection Reagent (Peqlab Biotechnology) according to the manufacturer’s
Stealth™ small interfering RNA (siRNA) duplex oligoribonucleotides for human MBTPS1 and
the “High GC negative control” were synthesized by Invitrogen. The sequence of MBTPS1
siRNA (+-strands) was as followed: 5-CAGAUGUGCUCUGGCAGAUGGGAUAUAUCCCA
UCUGCCAGAGCACAUCUG-3. HeLa cells grown on 3.5-cm plates were transfected twice at
48-h intervals using 125 pmol of single siRNA and 5 µl of Lipofectamine™ 2000 reagent
(Invitrogen). The cells were analyzed 96 h after the first transfection.
CHO-7 and SRD-12B cells were cultured in 1:1 mixture of DMEM and nutrient mixture F12
Ham (Invitrogen) with 5% FCS and penicillin/streptomycin. SRD-12B cells were maintained
with the addition of 5 mg/ml cholesterol, 1 mM sodium mevalonate, and 20 mM sodium oleate
(all from Sigma). Weekly selection with amphotericin B (Sigma) of SRD-12B cells were
performed as described elsewhere (27).
For analysis of media, cells were conditioned for 48 h in Opti-MEM® I (Invitrogen).
Metabolic labeling and immunoprecipitation. CHO-7 and SRD-12B cells were metabolically
labeled with 150 µCi/ml [35S]methionine (Hartmann Analytic) in methionine-free DMEM for
1 h. After removing the labeling medium, cells were either harvested or chased for 4 h in DMEM
containing 1% bovine serum albumin (BSA) and 0.25 mg/ml methionine. Cell extracts and media
were analyzed by immunoprecipitation of cathepsin Z followed by SDS-PAGE and fluorography
as previously described (26, 28).
Real-time PCR. Total RNA was isolated from cells using GeneJET™ RNA Purification Kit
(Fermentas). For cDNA synthesis 1 µg of total RNA and the High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems) were used according to the manufacturer’s instructions.
For real-time PCR, TaqMan™ Gene Expression Assays (Applied Biosystems) including pre-
designed probes and primer sets for human MBTPS1 (Hs00186886_m1), human ACTB
(Hs99999903_m1) and mouse Actb (Mm00607939_s1) were used. PCR reactions using
Maxima™ Probe qPCR Master Mix (Fermentas) were analyzed with the Mx3000P (Stratagene)
as described previously (28). The relative expression of MBTPS1 mRNA was normalized to the
level of ACTB mRNA in the same cDNA using the comparative CT method (2 –ΔΔCT).
Generation of Gnptab knock-in mice. Targeting vector construction and knock-in strategy has
been designed and performed by geneOway (Lyon, France). Ten kb of mouse genomic DNA
encompassing the murine Gnptab gene region surrounding exons 11 to 19 have been isolated
from a genomic DNA library (129/SvPas) and sequenced. The construction of the final targeting
vector comprising genomic sequences of the exon 12 to 18 with the inserted mutation c.3082insC
in exon 16, and the generation of the Gnptab knock-in mice will be described elsewhere (K.
Kollmann & T. Braulke, in preparation).
Histopathology and ultrastructural analysis. Mice were deeply anesthetized with an
intraperitoneal injection of sodium pentobarbital 100 mg/kg. A fixative solution of 4%
paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2–7.4 and 37°C was
perfused transcardially, and a tail clipping was removed for the genotypic analysis. The hind legs
of the perfused mice were immediately cut and the skin removed; they were submerged in a
solution of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2–
7.4, and stored overnight. Tissue was removed the next day and the tibiae were left in 10%
EDTA in phosphate-buffered saline, pH 7.4 (PBS) for 3 days. 100 µm thick Vibratom sections
were cut with a Vibratom (Leica VT 1000S). The sections were rinsed three times in 0.1 M
sodium cacodylate buffer (pH 7.2–7.4) and osmicated using 1% osmium tetroxide in cacodylate
buffer. Following osmication, the sections were dehydrated using ascending ethyl alcohol
concentration steps, followed by two rinses in propylene oxide. Infiltration of the embedding
medium was performed by immersing the pieces in a 1:1 mixture of propylene oxide and Epon
and finally in neat Epon and hardened at 60 °C. Semithin sections (0.5 µm) were prepared for
LM mounted on glass slides and stained for 1 min with 1% Toluidine blue. Ultrathin sections
(60 nm) were cut and mounted on copper grids. Sections were stained using uranyl acetate and
lead citrate. Thin sections were examined and photographed using an EM902 (Zeiss) electron
Cells were grown after selection for another two to three days on Aclar®33C discs in 24 well
plates purchased from Electron Microscopy Sciences (Germany). They were either fixed as
monolayers or harvested and spinned down, fixed, embedded in Epon, cut and observed as
Endocytosis and processing of arylsulfatase B. Recombinant human arylsulfatase B (ASB) was
kindly provided by Dr. M. Vellard (BIOMARIN, Novato, CA) and iodinated with IODO-GEN
(Pierce, Rockford, IL) and Na [125I] (Hartmann Analytic) to a specific activity of 6 µCi/µg.
CHO-7 and SRD-12B cells grown on 35 mm dishes were incubated with 0.7 ml of medium
containing 0.1% BSA and 0.25 µCi [125I]-ASB in the presence or absence of 10 mM mannose 6-
phosphate (M6P) for 20 min at 37°C. After removal of the media the cells were washed and
either harvested or chased in FCS-containing medium for 2 and 4 h. The cells were counted and
analyzed by SDS-PAGE and autoradiography.
Other methods. Preparation of cell extracts, enzymatic deglycosylation of proteins and SDS-
PAGE followed by western blot analysis were performed as recently described (27). The content
of M6P-containing proteins in cell extracts was analyzed by scFv M6P-1 western blotting as
described (18). For double immunofluorescence microscopy transfected BHK cells were grown
on glass coverslips for 16 h followed by treatment with 100 µg/ml cycloheximid for 2 h. The
cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 in
PBS. SRD-12B and CHO-7 cells were grown on glass coverslips and permeabilized with
500 µg/ml filipin (Sigma) in PBS after paraformaldehyde fixation. Cells were incubated for 16 h
with primary antibodies and 1 h with secondary antibodies conjugated to Alexa Fluor® 546 and
Alexa Fluor® 488. After three washes the cells were embedded in Mowiol. Fluorescence was
detected and images were obtained using a Leica DMIRE2 digital scanning confocal microscope
and ADOBE PHOTOSHOP software, respectively. The activities of the lysosomal enzymes
β-hexosaminidase, β-galactosidase, α-mannosidase and α-fucosidase in protein extracts and
media of cultured cells were determined as described previously (29, 30).
Statistical analysis. Statistical analysis was performed using unpaired two-tailed student’s test.
Fig. S1. Structural requirements for the cleavage of the α α/β β-subunit precursor. Three
independent experiments were performed with CHO-7 (A) and BHK cells (B, C). Cells were
transfected with construct 3 cDNA (wt) or P6 to P6’ alanine mutants surrounding the cleavage
site followed by β-subunit western blotting. Constructs with mutations R925A, L927A and
K928A showed strongly reduced amounts of the β-subunit indicating reduced cleavage both in
BHK and CHO-7 cells. The mitochondrial marker MnSOD was used as loading control.
Fig. S2. Intracellular localization of wildtype and mutant α α/β β-subunit precursor
construct 3. (A) BHK cells were transfected with construct 3 (wt) or with the non-cleavable
mutant R925A construct 3. Endo H treatment of cell extracts for 1 h increased the mobility of the
α/β-subunit precursor construct 3 to approximately the same extent as PNGase F, whereas the
N-linked sugars of the formed β-subunit were mostly Endo H-resistant. The R925A precursor
mutant is represented by a 120/110 kDa doublet. Endo H removed about half of the N-linked
oligosaccharides, implying that the α/β-subunit precursor reached the medial Golgi apparatus.
(B) Double immunofluorescense microscopy of BHK cells showed that most of transfected myc-
tagged full length construct 1, construct 3 (Fig. 1A) and the mutant R925A α/β-subunit precursor
construct 3 (green) is colocalized (yellow) with the cis-Golgi marker GM130 (red). Magnified
views of the indicated white rectangles are shown on the right. Scale bar, 20 µm.
Fig. S3. Histopathology and ultrastructural analysis of tibiae of wildtype and MLII mice.
Chondrocytes of the epiphyseal growth plate of 4 months old wildtype (wt) and mucolipidosis II
(MLII, Gnptabc.3082insC) mice are shown. Toluidin blue-stained semithin sections (left) show
strongly ballooned and disarranged chondrocytes in the MLII mice (arrows) filled with numerous
storage lysosomes shown by electron microscopy and indicated by red stars (right). Scale bars
left = 20 µm; right = 5 µm.
Fig. S4. Cytology and ultrastructural analysis of CHO-7 and SRD-12B cells. Upper panels
show semithin sections of cells grown on Aclar discs stained with Toluidin blue. Many cells of
the SRD-12B cultures show vacuoles and presumably storage lysosomes. In high power electron
micrographs huge numbers of storage material clustered around the cell body (N) and large
vacuoles are found in the SRD-12B cells whereas the cytoplasm of the CHO-7 cells is densely
filled with ribosomes, mitochondria (M) and other organelles. Scale bars upper panel = 20 µm;
middle and lower panel = 1 µm.
Fig. S5. Accumulation of cholesterol and bis(monoacylglycero)phosphate in SRD-12B cells.
Immunofluorescence microscopy of unesterified cholesterol stained with filipin (blue) and the
lysosomal marker Lamp2 (green) (A) or the lysophospholipid bis(monoacylglycero) phosphate
(BMP; green) (B) showed an accumulation of cholesterol in the SRD-12B cells. The cholesterol
is partially colocalized with Lamp2 and BMP. Magnified views of the indicated white rectangles
are shown on the right. Scale bars left = 15 µm.
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12 Download full-text
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