3
0 strokes of a Dounce homogenizer. Nuclei and debris were pel-
incubated with cross-linker at room temperature for 1 h. For
microsomal protein cross-linking, XLA or XLB were dried in
1.5 ml microtubes under air, dissolved in water, sonicated, and
stored at 20°C until use. Thawed cross-linker micelles (0.1 µg)
were preincubated with 2 mM GLTP and 13 µg GlcCer micelles
or water of equal volume at 37°C for 1 h. GLTP, cross-linkers,
leted at 1,000 g for 10 min at 4°C. The supernatant was collected
and centrifuged at 10,000 g for 10 min at 4°C. The supernatant
(crude microsomes) was collected, and protein concentration
measured. The protease inhibitor AEBSF was added to 0.1 mM,
and 200 g aliquots were stored at 80°C. This microsome prepa-
ration method is designed to retain cytosolic factors and minimize
their dilution and is a modification of that used by De Rosa et al.
and GlcCer samples were incubated with 1 mM MnCl , 1 mM
2
Mg(OAc) , 20 mM cacodylate pH 6.8, protease inhibitor cock-
2
(
9); DTT was omitted to avoid reduction of the cleavable disulfide
tail, 0.5 mM UDP-Gal, 0.25 mM CBE, and 100 g DU145 micro-
somes at 37°C for 1 h. Purified and microsomal proteins were
cross-linked with Spectroline Model EB-280C UV (302 nm)
from a distance of 5 cm for 15 min. Samples were analyzed by
SDS-PAGE, stained with Coomassie blue, or by Western blot
using SA-HRP.
bond in XLA and XLB.
LacCer synthase (LCS) assays
Detergent-containing assay to measure LCS activity. Triton X-100
0.3%) and 20 g C16:0 GlcCer, XLA or XLB were dried
(
under nitrogen, suspended, and sonicated in 20 mM cacodyl-
ate, pH 6.8, 10 mM MnCl , and 1 mM MgCl and incubated with
2
2
3
1
3
30 g DU145 microsomes and 0.5 Ci H-UDP-Gal at 37°C for
h (24).
RESULTS
Design and synthesis of 2A-GlcCer XL (XLA and XLB)
photoactivatable cross-linkers
Detergent-free assay to assess GlcCer translocation. The detergent-
free radiolabeled LCS assay is a modification of that of Chatterjee
and Castiglione (24). Bovine GlcCer (20 g) was dried and soni-
cated in water, incubated with 20 mM cacodylate, pH 6.8, 10 mM
MnCl and 1 mM MgCl , 2 M GLTP, 5 M ATP, 120 g intact
The 2A-GlcCer XL was designed to closely mimic native
GlcCer with two hydrophobic chains for membrane asso-
ciation, a cleavable disulfide bond, photoactivatable aryl
azide in the proximity to the head group, and a biotin tag
for affinity isolation. An overview of 2A-GlcCer XL synthe-
sis is shown in Fig. 1. GlcCer was deacylated to lyso-GlcCer,
desalted on a C-18 reverse phase silica gel column, and pu-
rified. The amino function of 2-aminohexadecanoic acid
was protected with t-BOC, which increases yield of the sub-
2
2
3
DU145 microsomes, and 0.5 Ci H-UDP-Gal (100 l final vol-
ume) at 37°C for 3 h.
For both LCS assays, phospholipids were saponified at room
temperature for 2 h with 1 M NaOH in methanol. Each reaction
was neutralized with equal normal HCl(aq) and 23 mM NH OAc.
4
Chloroform was added to form a Folch partition (CHCl3/
CH OH/water, 2:1:0.6, v/v/v), the GSL-containing lower phase
3
was washed with theoretical upper phase (CHCl /CH OH/water, sequent BOP reaction by preventing polymerization of the
3
3
1
:47:48, v /v/v), a nd t he l ower p hase w as e xtracted a nd d ried u n-
2-amino fatty acid. TLC staining with ninhydrin was used to
monitor t-BOC protection reaction progression (Fig. 2A).
der nitrogen. Samples were dissolved in 20 µl 2:1 CHCl /CH OH,
and half was separated by TLC CHCl /CH OH/water (65:25:4,
v/v/v), along with a standard, C-Gal-labeled GSLs from vero
cells. The TLC plate was sprayed with En3Hance and exposed to
film at 80°C.
3
3
3
3
2-Aminohexadecanoic acid contains both d and l forms,
1
4
which appears to run as two distinct bands when separated
by TLC. After t-BOC protection the d, l isomers may have
closely resolved to appear as one band. Product was puri-
fied on silica column and was confirmed by negative TOF
mass spectrometry to have the expected mass of t-BOC pro-
tected 2-aminohexadecanoic acid [2-((tert-butoxycarbonyl)
amino)hexadecanoic acid], 371.3 amu. Lyso-GlcCer was
coupled to 2-((tert-butoxycarbonyl)amino)hexadecanoic
acid to produce the product t-BOC-protected 2A-GlcCer.
Reaction progression was monitored by TLC compared
with C16:0 GlcCer analog and lyso-GlcCer by orcinol
staining (Fig. 2B). t-BOC-protected 2A-GlcCer was deprot-
ected, the two products (A and B) were purified and
compared by TLC (Fig. 2C), and 2A-GlcCer A and B were
confirmed as isomers by positive TOF mass spectrometry
with a mass of 714.6 amu.
Diastereomers are not usually so well resolved by TLC
(Fig. 2C). Therefore, the amino group on the fatty acid
moiety of the 2A-GlcCer analogs was acetylated, to probe
whether the TLC separation of the two isomers is due to
differential intramolecular hydrogen bonding. After acet-
ylation, the separation of the two isomers was much re-
duced, suggesting that polarity of isomer A is due to an
NH intramolecular hydrogen bond (Fig. 2D). Both acety-
lated isomers A and B had an expected mass of 756.6 amu.
2A-GlcCer A and B analogs were separately coupled to
sulfo-SBED to synthesize the biotinylated cross-linkers
NBD-GlcCer LCS assay. For synthesis of NBD-GlcCer, lyso-
GlcCer (2.5 mg) and NBD-X SE (3.2 mg) were dissolved in 1.1 ml
of DMF/TEA (9:1, v/v) and stirred at room temperature for 3 h
(25). NBD-GlcCer (80% conversion) was purified by silica gel col-
umn chromatography and stored at 20°C in chloroform. For
the detergent-free NBD-LCS assay, the LCS assay is a modification
of the method of Chatterjee and Castiglione (24). Primarily in-
tact, right-side-out microsomes were used. Bovine NBD-GlcCer
(1 g), and XLA or XLB (20 g) were dried under nitrogen, dis-
solved in water, sonicated, and diluted to a total volume of 100 l
containing 1 mM MnCl , 1 mM Mg(OAc) , 20 mM cacodylate pH
2
2
6.8, protease inhibitor cocktail (50 M AEBSF, 40 nM aprotinin,
25 M bestatin, 75 nM E-64, 1 M leupeptin, 0.5 M pepstatin A),
0.5 mM UDP-Gal, 0.25 mM CBE, and 100 g DU145 microsomes.
Enzyme reactions were incubated at 37°C as indicated and stopped
by addition of 1 ml of CHCl /CH OH ( 2:1, v /v) a nd 1 00 l water
for Folch partition as above. Half of each sample was separated by
TLC CHCl /CH OH/water ( 80:20:2, v /v/v). T he d ried p late w as
imaged using a Typhoon FLA 9500 Fluorescent imager (473 nm
excitation wavelength, 250 V, 50 µm pixel size).
3
3
3
3
Cross-linking assay
For purified protein cross-linking, XLA or XLB were dried,
dissolved in 2 µl of ethanol (0.75 µM final concentration), incu-
bated at 37°C for a few minutes, and then diluted in warm TBS.
Equal mass of GLTP (2 mM), gelatin, VTB, and CTB in TBS were
1730
Journal of Lipid Research Volume 57, 2016
Budani, Monique
