F. Preugschat et al. / Archives of Biochemistry and Biophysics 564 (2014) 156–163
157
nucleotides) that has been used to characterize and isolate
alternative substrates and inhibitors of these enzymes [14–17].
CD157 (often referred to as; bone marrow stromal antigen
BST-1, NAD+ nucleosidase or cyclic ADP-ribose hydrolase 2) was
initially cloned by differential screening of synovial fluid from
patients with a severe form of rheumatoid arthritis [18–19]. The
gene for this enzyme is located on human chromosome 4
media was concentrated using 10 kDa cutoff Pall filters. The con-
centrate was buffer-exchanged with 20 L of 25 mM Hepes pH 7.5,
250 mM NaCl, and was re-concentrated to 2 L final volume. This
concentrate was initially purified on a nickel Sepharose column
and eluted using imidazole. The N-terminal histidine tag was
removed by digestion with TEV protease and the enzyme was then
reapplied to a nickel Sepharose column. The flow-through fraction
was collected and applied to a superdex 75 sizing column, equili-
brated with buffer. Peak fractions were concentrated and stored
frozen. Peptide sequencing of trypsin-digested hCD157 fragments
confirmed the identity of the purified protein.
(
[
4p15.33) and is a product of gene duplication of the CD38 locus
20]. The exon structure and gene regulatory elements are highly
conserved between CD38 and CD157. Because hCD157 is a younger
gene than hCD38, the human protein is 78–89% identical to the
mouse and rat homolog, respectively. Despite their common ances-
try there are notable differences. CD38 is an integral membrane
protein, whereas CD157 is associated with the cellular membrane
CD38 (E226Q) protein purification
through a C-terminal glycophosphatidylinositol (GPI) linkage [20].
+
10 L of conditioned media from Pichia pastoris – expressing
human CD38 (N100D, N164A, N209D, N219D, E226Q with a
N-terminal histidine tag and TEV protease cleavage site) was applied
to a nickel Sepharose column. The column was washed with buffer,
CD38 is a powerful hydrolytic catalyst (NAD
k
cat/K
M
value of
ꢀ1
ꢀ1
+
+
1
0
lM
s
), whereas CD157 is an ineffective (NAD and NGD
ꢀ
1 ꢀ1
kcat/K
M
value of 0.2–1 mM
s ) hydrolytic catalyst [15,21–22].
In summary CD157 is a protein with novel, but related biochemical
properties.
(
25 mM Hepes pH 7.5, 250 mM NaCl, 50 mM imidazole) and
protein was eluted with buffer (25 mM Hepes pH 7.5, 250 mM
NaCl, 250 mM imidazole). The histidine tag was removed by
digestion with TEV protease and was chromatographed on a
superdex 75 sizing column. Fractions containing CD38 were pooled
and passed over a nickel Sepharose column to remove any CD38
containing the histidine tag. Flow-through fractions containing
CD38 were pooled and concentrated to 5 mg/mL. Sequence
identity with CD38 was confirmed by LC/MS amino acid sequence
analysis of trypsin-digested peptides. The enzyme was >95% pure.
Nicotinamide riboside (NR) was initially characterized in the
1
940s and was recently shown to function as a precursor vitamin
+
for eukaryotic NAD biosynthesis [23–24]. Intracellular phosphor-
ylation of NR produces NMN, an intermediate that is then subse-
+
+
quently adenylated to form NAD in the NAD salvage pathway.
+
Because exogenously-added NR raises intracellular NAD levels in
mammalian cells (2–3-fold) NR could be a dietary supplement that
elevates NAD levels [25]. It is possible that inhibition of NR catab-
+
olism may be a potential mechanism for increasing cellular NAD
levels.
During the course of characterizing the biochemical selectivity
of CD38 inhibitors, we discovered that hCD157 prefers NR over
Crystallographic analyses of a binary complex of NMN with the hCD38
catalytic mutant E226Q
+
ꢀ1 ꢀ1
NAD as a substrate for hydrolysis (kcat/K
M
value of 17
lM
s ).
Herein, we present evidence that the enzyme uses covalent catal-
ysis with formation of a ribosylated enzyme intermediate. In the
absence of nicotinamide, the level of ribosylated enzyme is high
at low levels of NR. Thus the enzyme is effectively capturing the
ribose portion of NR at very low levels of NR. The proposed ribosy-
lated intermediate is relatively long-lived, suggesting that NR may
function to activate hCD157 as a receptor. Alternative substrates
Human CD38 E226Q in 25 mM Hepes pH 7, 200 mM NaCl was
concentrated to approximately 7 mg/mL. 5 mM NMN was added
to the protein and the complex was incubated on ice for 1 h. Crys-
tals were grown by hanging drop vapor diffusion in 24-well Linbro
trays at room temperature in 23% PEG3350, 0.1 M BTP, pH 8.5.
Crystals were harvested after 1 day and flash frozen in PFO prior
+
to data collection. Data were collected in house on a Sat944 detec-
(
analogs of NR) or antagonists (inhibitors) of hydrolytic function
tor and Rigaku FRE rotating anode generator. Data were processed
using HKL2000 [27]. The structure was solved by molecular
replacement using MOLREP [28] and 1YH3 (human CD38 extracel-
lular domain) as a starting model [29]. The structure was rebuilt
and refined using CCP4 [30] and Coot [31]. Coordinates have been
deposited in the RCSB database as PDB code (4OGW).
may prove to be useful therapeutic agents for the treatment of a
number of human diseases.
Materials and methods
Materials
+
Assay of hydrolytic activity
NAD , NMN, nicotinamide, alkaline phosphatase, phosphate
buffered saline (PBS), Hepes, nicotinic acid, and EDTA were from
Sigma Chemical Co. hCD157 (catalog number 4736-AC, lot #
RLZ0311071) was purchased from R & D Systems, Minneapolis,
MN or from Reprokine Ltd., Rehovot, Israel (catalog number RKQ
Hydrolysis of the nicotinamide ribose bond was monitored
through a decrease in absorbance of the nucleos(t)ide [32]. The
values of the difference extinction coefficients used were:
ꢀ1
ꢀ1
ꢀ1
ꢀ1
D
e
262 = ꢀ1.4 mM cm
for NR,
D
e
280 = ꢀ1.2 mM cm
for
1
0588). NR was purchased from Toronto Research Chemicals.
+
ꢀ1
ꢀ1
NMN and NAD ,
D
e280 = ꢀ1.7 mM cm for NAR.
Purification of recombinant proteins
HPLC analyses of hCD157-catalyzed NR hydrolysis
Our initial characterization of two commercially available
hCD157 proteins revealed biochemical differences between the
proteins. Due to these differences we decided to clone, express
and purify hCD157. hCD157 amino acids 29–303 with a N-terminal
histidine tag and a TEV cleavage site were cloned into a Bacmam
expression vector for Bacmam virus production [26]. Bacmam
virus was used to transfect CHO cells and cell culture supernatants
were harvested 72 h post-infection. 10 L of conditioned cell culture
Standards and reaction products were analyzed on a Agilent
1100 series HPLC, using a 10 cm reverse phase C18 column (Phe-
nomenex). 50 mM ammonium acetate, pH 7.0 was used for mobile
phase A. 33 mM ammonium acetate, pH 7.0 in 70% acetonitrile was
used for mobile phase B. A gradient of 0–100% mobile phase B was
run over a 10 min time-course. Analytes were detected using a
diode array spectrophotometer.