Tài liệu Báo cáo khoa học: Restricted localization of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase at the neuromuscular junctions – contribution and expression from motor neurons doc

Restricted localization of proline-rich membrane anchor
(PRiMA) of globular form acetylcholinesterase at the
neuromuscular junctions – contribution and expression
from motor neurons
K. Wing Leung, Heidi Q. Xie, Vicky P. Chen, Mokka K. W. Mok, Glanice K. Y. Chu, Roy C. Y. Choi
and Karl W. K. Tsim
Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, China

Keywords
acetylcholinesterase; molecular form;
muscle fiber type; neuromuscular junction;
proline-rich membrane anchor
Correspondence
K. W. K. Tsim, Department of Biology,
The Hong Kong University of Science and
Technology, Clear Water Bay Road,
Kowloon, Hong Kong SAR, China
Fax: +852 2358 1559
Tel: +852 2358 7332
E-mail:
(Received 21 November 2008, revised 11
March 2009, accepted 25 March 2009)
doi:10.1111/j.1742-4658.2009.07022.x

The expression and localization of the proline-rich membrane anchor
(PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), were studied at vertebrate neuromuscular junctions.
Both muscle and motor neuron contributed to this synaptic expression
pattern. During the development of rat muscles, the expression of PRiMA
and AChET and the enzymatic activity increased dramatically; however,
the proportion of G4 AChE decreased. G4 AChE in muscle was recognized
specifically by a PRiMA antibody, indicating the association of this enzyme

with PRiMA. Using western blot and ELISA, both PRiMA protein and
PRiMA-linked G4 AChE were found to be present in large amounts in
fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slowtwitch muscle (e.g. soleus). These results indicate that the expression level
of PRiMA-linked G4 AChE depends on muscle fiber type. In parallel, the
expression of PRiMA, AChET and G4 AChE also increased in the spinal
cord during development. Such expression in motor neurons contributed to
the synaptic localization of G4 AChE. After denervation, the expression of
PRiMA, AChET and G4 AChE decreased markedly in the spinal cord, and
in fast- and slow-twitch muscles.

Acetylcholinesterase (AChE; EC 3.1.1.7) plays a crucial role in terminating the synaptic transmission by
hydrolyzing the neurotransmitter acetylcholine at the
neuron-to-neuron synapses in the central nervous system and at the neuromuscular junctions (NMJs) in the
peripheral nervous system. AChE exists in different
molecular forms. The formation of these molecular
forms depends on alternative splicing in the 3¢ region
of the primary transcript [1], which generates the
AChER (‘readthrough’), AChEH (‘hydrophobic’) and
AChET (‘tailed’) subunits, containing the same catalytic domain but different carboxyl termini [1]. In

mammals, the AChER variant produces a soluble
monomer that is up-regulated in the brain during
stress [2]; the AChEH variant produces a glycosylphosphatidylinositol-linked dimer and is expressed in blood
cells; the AChET variant is the only subunit expressed
in the brain and muscle. The AChET subunits form
nonamphiphilic tetramers with a collagen tail as asymmetric AChE (A4, A8 and A12) in muscle. In addition,
the AChET variant produces monomers (G1), dimers
(G2) and tetramers (G4). The amphiphilic tetramer
(G4) is linked with a proline-rich membrane anchor
(PRiMA) as a globular form of AChE (PRiMA-linked

Abbreviations
AChE, acetylcholinesterase; AChR, acetylcholine receptor; BChE, butyrylcholinesterase; ChAT, choline acetyltransferase; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei; NMJs, neuromuscular junctions;
PRiMA, proline-rich membrane anchor; SNAP-25, synaptosomal-associated protein 25.

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G4 AChE) in brain and muscle [3–5]. Two PRiMA
isoforms (PRiMA I and PRiMA II) are generated
from the PRiMA gene by alternative splicing. PRiMA I contains a longer C-terminal cytoplasmic
domain than does PRiMA II [6].
Although asymmetric AChE is the predominant
species at NMJs and its appearance in muscle coincides with the establishment of neuromuscular contacts
during development and regeneration [7,8], G4 AChE
also exists in muscles. Several studies have revealed
that the level of G4 AChE is controlled by the
dynamic activity of skeletal muscles. The transcriptional regulation of PRiMA is down-regulated during
myogenic differentiation and under the influence of
innervation [9]. In line with the transcriptional expression of PRiMA, the proportion of G4 AChE decreases
during myogenic differentiation and innervation [1,9].
In mammals, fast-twitch muscles contain a large
amount of G4 AChE, whereas slow-twitch muscles
contain a much smaller amount [10].
The expression of different AChE forms at NMJs

raises the question of whether the synaptic enzyme is
produced by muscle, nerve or both under different
physiological states. Both asymmetric and globular
forms of AChE are known to be produced by muscle
cells [11,12], and the presynaptic motor nerve terminals
synthesize and secrete AChE at NMJs [13,14]. The predominant form of AChE expressed by motor neurons
in chick spinal cord is G4 AChE [15].
In this article, we analyze the expression and localization of the PRiMA I-linked G4 form of AChE in rat
muscles and motor neurons. We prepared an antibody

K. W. Leung et al.

against the cytoplasmic domain of PRiMA I, which
allowed us to show that PRiMA-linked G4 AChE is
localized at NMJs in both presynaptic nerve terminals
and postsynaptic muscle fiber. It is expressed by motor
neurons in the rat spinal cord: this expression
increased during development, but decreased after
denervation. These data show that both presynaptic
motor neuron and postsynaptic muscle fiber contribute
to the synaptic expression of PRiMA-linked G4 AChE
and illustrate its temporal and spatial expression at
NMJs.

Results
Regulation of G4 AChE and PRiMA during muscle
development
A rabbit polyclonal antibody against the C-terminus
of PRiMA I was generated. To validate the PRiMA
antibody, a full-length mouse PRiMA cDNA (corresponding to PRiMA I unless specified) and a C-terminal truncated mutant (PRiMADC-term) cDNA, both

tagged with a FLAG epitope, were transfected into
HEK293T cells. In western blot analysis, a FLAG
antibody recognized both PRiMA and PRiMADC-term
with protein bands of approximately 20 and 16 kDa,
respectively: these protein bands corresponded to the
predicted size of the recombinant proteins (Fig. 1A).
The PRiMA antibody, however, recognized only
the full-length PRiMA, but not the truncated
PRiMADC-term construct. In addition, the recognition
was fully blocked by pre-incubation of the PRiMA

Fig. 1. The specificity of the PRiMA antibody. (A) Protein samples (40 lg) of HEK293T cells expressing FLAG-PRiMA or FLAG-PRiMADC-term
were analyzed by 12% SDS–PAGE. Both PRiMA and FLAG antibodies (Ab) were used to label the PRiMA proteins. In the blocking experiment,
excess amounts of recombinant PRiMA antigen (Ag) (from residues 114 to 153) at 5 lgỈmL)1 were pre-incubated with the PRiMA antibody
(0.5 lgỈmL)1) for 4 h at 4 °C before it was used for western blotting. (B) Transfected HEK293T cells were stained with PRiMA or FLAG antibody
as described in Materials and methods. Bar, 10 lm.

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K. W. Leung et al.

antibody with the antigen, i.e. the PRiMA I
C-terminal peptide (Fig. 1A). In the immunocytofluorescent staining of transfected fibroblasts, the PRiMA
antibody also recognized FLAG-tagged PRiMAexpressing cells (Fig. 1B). In contrast, FLAG-tagged
PRiMADC-term-expressing cells were not recognized by
the antibody. As a positive control, FLAG antibody
was used; it recognized both full-length and truncated

PRiMA in protein detection and immunostaining
(Fig. 1A,B). Such recognition could not be blocked by
pre-incubation with PRiMA antigen. These results
clearly indicate the specificity of the PRiMA antibody
in recognizing the cytoplasmic domain of PRiMA I.
According to Perrier et al. [6], two splicing variants
of PRiMA mRNAs are generated from the PRiMA
gene to produce different proteins (PRiMA I and
PRiMA II; Fig. 2A). PRiMA I mRNA, which possesses exons 4 and 5, produces a 40-residue-long intra-

Proline-rich membrane anchor at neuromuscular junctions

cellular cytoplasmic tail, whereas PRiMA II mRNA,
which possesses exons 4, 4b and 5, encodes a shorter
intracellular motif. These two PRiMA isoforms may
be distinguished by RT-PCR using primers flanking
exons 4 and 5. In rat muscles, PRiMA I was found to
be present, whereas PRiMA II was barely detectable
(Fig. 2B). For precise quantification, we used real-time
PCR with the same set of primers. In agreement with
the absence of PRiMA II in muscle, all the amplified
products revealed by real-time PCR corresponded to
PRiMA I. The mRNA level of PRiMA I was up-regulated gradually in the early postnatal stages and dramatically in the adult stage (Fig. 2B). Meanwhile, the
level of AChET mRNA increased gradually from the
early postnatal stage to the adult. Using PRiMA antibody, the PRiMA protein was detected in the muscles
of embryonic rats; its level increased after postnatal
day 10 to the adult (Fig. 2C). As reported previously,

Fig. 2. Developmental profiles of PRiMA, AChET and G4 AChE in skeletal muscles. (A) Splice variants of PRiMA mRNAs (PRiMA I and II) are
illustrated. PRiMA II contains an additional exon 4b. Arrows show the location of primers used for qualitative and real-time PCR analyses. (B)

Total RNAs were extracted from rat leg muscles at different developmental stages to perform RT-PCR for PRiMA I (145 bp) PRiMA II
(302 bp) and AChET (671 bp). Adult rat brain served as a positive control. One representative result is shown (top). The bottom panel shows
the results of real-time PCR analysis of the mRNA expression of PRiMA I and AChET. (C) Samples of extracts from the lower leg muscles
of rat (birth to adult stage) containing 40 lg of protein were loaded per lane for western blotting (top). The levels of PRiMA and AChET
proteins were determined. GAPDH served as a loading control. The bottom panel shows the quantified data of protein bands. AChE activity
was determined by the Ellman assay. (D) Samples of extracts from rat leg muscles containing equal amounts of AChE activity were loaded
on sucrose density gradients. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown. (E) The specific activity of
G4 AChE was quantified at different developmental stages. Samples of muscle extracts at different developmental stages containing 600 lg
of protein were loaded on sucrose density gradients. The peak area corresponding to G4 AChE activity was determined. The results are
expressed as the ratio to the value obtained at E21 (basal), and are shown as means ± standard error of the mean (SEM), n = 4.

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AChET protein and AChE enzymatic activity increased
during muscle development (Fig. 2C). With regard to
the AChE molecular form, the AChE G1 and G4 forms
were predominant in embryonic muscles (Fig. 2D). In
mature muscles, the relative proportion of the G1 and
G4 forms was reduced and the asymmetric form of
AChE (A12) was increased (Fig. 2D). In order to quantify the relative amount of PRiMA-linked G4 AChE in
developing muscle, protein extracts at different developmental stages were analyzed by sedimentation in sucrose
density gradients. The proportion of G4 AChE was
determined from the peak area, relative to the area of

the entire sedimentation profile, and its activity was
given by the product of this proportion with the total
AChE activity. The amount of G4 AChE in muscle
increased twofold from birth to adult (Fig. 2E).
PRiMA-linked G4 AChE therefore increased during
muscle development.
Expression of PRiMA-linked G4 AChE in
fast-twitch and slow-twitch muscles
In order to investigate the expression level of PRiMA
and PRiMA-linked G4 AChE in different muscle fiber
types, fast-twitch (tibialis) and slow-twitch (soleus)
muscles from adult rats were collected and analyzed. In
western blotting, PRiMA protein was detected in both
tissues, but its level was about threefold lower in the
soleus than in the tibialis (Fig. 3A). The relative abundance of PRiMA-linked G4 AChE was determined by
ELISA using our PRiMA antibody. Equal amounts of
AChE activity were loaded onto an ELISA plate precoated with serial dilutions of PRiMA antibody. The
retained AChE enzymatic activity, corresponding to
PRiMA-linked G4 AChE, was measured after washing.
We found larger amounts (over twofold) of PRiMAlinked G4 AChE in the tibialis than in the soleus
(Fig. 3B). The higher expression of G4 AChE in the
tibialis was further confirmed by sucrose density gradient analysis. The PRiMA antibody was able to deplete
the G4 form of AChE in the tibialis, but this was not
obvious in the soleus (Fig. 3C). In all cases, the brain
enzyme was used as a control.
We analyzed the localization of PRiMA in sections
of tibialis and soleus muscle by immunohistofluorescence. NMJs were visualized by labeling the postsynaptic
acetylcholine
receptor
(AChR)

with
a-bungarotoxin (shown in red or pseudo-blue) and the
presynaptic nerve terminal with synaptotagmin (SV48;
shown in red) in both types of muscle (Fig. 4). PRiMA
(shown in green) was expressed at the NMJs, and its
distribution was wider than that of AChE and AChR,
extending into a peri-junctional zone where neither
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Fig. 3. Expression of PRiMA and G4 AChE in different muscles. (A)
Samples of extracts from adult rat soleus and tibialis containing
40 lg of protein were loaded per lane for western blotting of PRiMA
protein. Adult rat brain served as a positive control. The bottom panel
shows the quantification of PRiMA protein. The results are
expressed as the ratio to soleus (basal) equal to unity; means ± standard error of the mean, n = 4. (B) The relative amount of PRiMAlinked G4 AChE was quantified by ELISA. Tissue lysates from rat
brain, tibialis and soleus containing equal AChE activities were loaded
onto an ELISA plate precoated with serial dilutions of PRiMA antibody for 2 h. The retained AChE activity was determined. (C) For
immunodepletion, 1 mL samples of extracts from adult rat brain, tibialis and soleus were incubated with PRiMA antibody (10 lgỈmL)1)
and protein G-agarose before sucrose density gradient analysis.
AChE activity was plotted as a function of the S value, estimated
from the position of the sedimentation markers. Enzymatic activities
are expressed in arbitrary units, and representative sedimentation
profiles are shown.

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Proline-rich membrane anchor at neuromuscular junctions

Fig. 4. Localization of PRiMA and AChE at
NMJs. Sections from adult rat tibialis (top)
and soleus (bottom) muscles were triple
stained with rhodamine-conjugated or Alexa
647-conjugated a-bungarotoxin (red or
pseudo-blue) for postsynaptic AChR, antiAChET (pseudo-blue), anti-synaptotagmin
(SV48; red) for presynaptic nerve terminal
and anti-PRiMA (green), and examined by
confocal microscopy. Merged images of
AChR ⁄ SV48 and PRiMA are shown on the
right. Representative images are shown,
n = 4. Bar, 20 lm.

Fig. 5. Developmental evolution of PRiMA and G4 AChE in the spinal cord. (A) Total RNAs were extracted from spinal cord at different
developmental stages for detection of transcripts encoding PRiMA I (145 bp), PRiMA II (302 bp) and AChET (671 bp). Representative results
are shown. (B) Samples of extracts of rat spinal cord (from birth to adult stages) containing 40 lg of protein were loaded per lane for western blotting. PRiMA and AChET proteins were determined. GAPDH served as a loading control. (C) Quantification of proteins (from B) and
AChE activity during development. (D) One milliliter samples of extract from adult rat spinal cord, with and without depletion by the PRiMA
antibody (as in Fig. 3C), were analyzed by sucrose density gradients. AChE activity was plotted as a function of the S value, estimated from
the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles
are shown. (E) G4 AChE specific activity in the spinal cord at different developmental stages was quantified as in Fig. 2E. The results are
expressed as the ratio to the value obtained at P1 (basal) equal to unity; means ± standard error of the mean (SEM), n = 4.

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AChE nor AChR was present in either muscle fiber
type. However, the precise localization of PRiMA has
yet to be determined.
Presence of PRiMA-linked G4 AChE in motor
neurons
At NMJs, AChE may originate from the muscle fiber
and ⁄ or from the motor neuron. In order to examine the
presence of PRiMA and PRiMA-linked G4 AChE, rat
spinal cords were collected at early postnatal and adult
stages. Qualitative PCR indicated that both PRiMA I
and II transcripts were expressed in the spinal cord: the
PRiMA I transcript decreased slightly after birth, but
increased dramatically thereafter and was the predominant form in the adult, the PRiMA II transcript first
increased but disappeared in the adult (Fig. 5A). As a
result of the absence of a specific primer for PRiMA I,
the expression level of the PRiMA I transcript could
not be analyzed by real-time PCR. The PRiMA I protein level in the spinal cord, determined in western blots
with the PRiMA antibody (recognizing the cytoplasmic
domain of PRiMA I), increased after birth, as did
AChE (Fig. 5B,C). This was consistent with an increase
in total AChE activity (Fig. 5C) and with the observation that G4 was the predominant form of the enzyme in
the adult spinal cord (Fig. 5D). The majority of
G4 AChE was associated with PRiMA I, as more than
70% was immunoprecipitated with the PRiMA antibody. The relative amount of G4 AChE determined
from sedimentation profiles allowed us to evaluate its
activity: the specific activity of G4 AChE per milligram
of protein reached a plateau in the spinal cord about
10 days after birth (Fig. 5E).

To determine the origin of AChE in the spinal
cord, the lumbar region of the spinal cord was
sectioned and stained with the PRiMA antibody. The
label was mostly present in the ventral horn
(Fig. 6A). As expected, PRiMA was detected in
AChE-positive cells in the ventral horn (Fig. 6B).
These PRiMA-stained cells were motor neurons, as
shown by their reactivity with an anti-choline acetyltransferase (anti-ChAT) antibody. This identification
was further supported by double staining of neuronal
nuclei with a neuronal marker (NeuN). In contrast,
no PRiMA was found in glial cells that were labeled
specifically with an antibody against glial fibrillary
acidic protein (GFAP) (Fig. 6B). These results clearly
show that PRiMA is synthesized by motor neurons in
the spinal cord.
Although motor neurons are able to synthesize
PRiMA and produce G4 AChE, the restricted localization of PRiMA-linked G4 AChE at NMJs could still
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Fig. 6. Motor neurons in the spinal cord express PRiMA. (A) Schematic diagram showing the lumbar region of the spinal cord (left).
The dorsal horn and ventral horn are indicated. The right panel
shows PRiMA staining in the lumbar region on the same scale at
low magnification. The boxed area is shown at higher magnification
in (B). Bar, 100 lm. (B) Spinal cord sections were double stained
with anti-PRiMA (green) and with anti-AChET (red), anti-ChAT (red),
anti-NeuN (red) or anti-GFAP (red), and examined by confocal
microscopy. PRiMA was co-localized with AChET, ChAT and NeuN,
but not with GFAP. Representative images are shown, n = 4. Bar,
20 lm.

be derived from three sources: muscle, Schwann cells
and ⁄ or motor neurons. In order to determine the localization of PRiMA-linked G4 AChE, sections of tibialis
muscle were triple stained for PRiMA, SV48 and
AChR. The staining of PRiMA was coincident with
that of SV48, rather than with that of AChR (Fig. 7,
left panel). Similar results were obtained with another

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Proline-rich membrane anchor at neuromuscular junctions

Fig. 7. Presynaptic localization of PRiMA at NMJs. Adult rat tibialis sections were triple stained with Alexa 647-conjugated a-bungarotoxin
(pseudo-blue), anti-synaptotagmin (SV48; red) or anti-SNAP-25 (red) antibodies, and anti-PRiMA (green), and examined by confocal microscopy. Merged images allow a comparison of PRiMA with presynaptic markers (PRiMA + SV48 ⁄ SNAP-25) and a postsynaptic marker (PRiMA + AChR). The distribution of PRiMA overlapped with that of SV48 and SNAP-25. Representative images are shown, n = 4. Bar, 20 lm.

presynaptic marker, synaptosomal-associated protein 25 (SNAP-25): PRiMA also showed a better
co-localization with SNAP-25 than with AChR
(Fig. 7, right panel). Such overlapping of PRiMA
staining with presynaptic molecules indicates that
PRiMA at NMJs is mainly provided by motor
neurons.
Innervation regulates the expression of
PRiMA-linked G4 AChE in the spinal cord
The expression of PRiMA and the pattern of AChE
molecular forms in muscles are known to be modified
by denervation [7]. In order to determine whether
PRiMA expression in the spinal cord was regulated by
a retrograde influence of the muscle, a portion of the

sciatic nerve was surgically removed. After 7 days, we
examined the expression of PRiMA in both spinal
cord (lumbar region) and tibialis muscles by real-time
PCR analysis: PRiMA mRNA (PRiMA I) was not
modified significantly in the tibialis, but was reduced
by over 60% in the spinal cord (Fig. 8A). In contrast,
the mRNA level of AChET was decreased in both the
spinal cord and tibialis when compared with that of
the sham-operated control (Fig. 8A). At the protein
level, western blot analyses showed that PRiMA and
AChET were reduced by about 50% after denervation
in both tissues (Fig. 8B). This is consistent with a
decrease in AChE enzymatic activity of about 50% in
the spinal cord and tibialis muscle (Fig. 8B). Sucrose
density gradient analyses showed a significant reduction of G1 and G4 forms in the spinal cord and of G1,
G4 and A12 forms in the tibialis (Fig. 8C). Thus,
denervation induced a decrease in PRiMA and
G4 AChE in the spinal cord and muscle.

To investigate the contribution of the motor neuron
to PRiMA-linked G4 AChE at NMJs, we analyzed the
effect of denervation on the localization of PRiMA.
The NMJs of the denervated tibialis and sham-operated muscle were stained for PRiMA, together with
the postsynaptic marker AChR (shown in pseudo-blue)
and a presynaptic marker SV48 (shown in red). Presynaptic labeling essentially disappeared at the denervated NMJs and PRiMA labeling was considerably
reduced. This suggests that a significant proportion of
PRiMA was provided by the presynaptic motor
neuron (Fig. 8D). However, a small amount of
PRiMA could still be detected in the denervated
muscles, possibly of muscle origin.

Discussion
The muscles of mice in which the PRiMA gene is inactivated contain essentially no G4 AChE, suggesting
that this enzyme form is entirely associated with
PRiMA. Our results show that G4 AChE is, indeed,
largely immunoprecipitated with a PRiMA antibody.
However, a fraction of G4 AChE was not
immunodepleted (Fig. 3C), even when the amount of
antibody was increased or with a second round of
immunodepletion (not shown). The interaction of this
fraction with the antibodies may be prevented by the
presence of partner(s) associated with the C-terminal
region of PRiMA. In addition, no G4 AChE was
found in muscles of PRiMA knockout mice, implying
that all G4 AChE in muscle is linked with the membrane-anchoring protein PRiMA. During muscle development, the amount of PRiMA-linked G4 AChE
progressively increased from birth to the adult stage.
In addition, the expression of PRiMA and G4 AChE

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Fig. 8. Denervation reduces the expression of PRiMA and G4 AChE in the spinal cord and in muscles. (A) The sciatic nerve was sectioned
to examine the effect of muscle on the expression of PRiMA in motor neurons. After 7 days, tibialis and spinal cord were collected for analysis. The mRNA levels of denervated muscles (Den) corresponding to PRiMA (top) and AChET (bottom) were determined by PCR and normalized to those of control (sham-operated) muscles. (B) Samples of extracts from control and denervated muscles containing 50 lg of protein
were loaded per lane for the western blotting of PRiMA and AChET. GAPDH served as a loading control. The bottom panel shows the ratios

of AChE enzymatic activity after nerve section to control values. The results are expressed as the ratio to control values (sham-operated)
equal to unity; means ± standard error of the mean (SEM), n = 3. (C) Effect of nerve section on AChE molecular forms in the spinal cord
and tibialis muscles. Samples containing equal amounts of protein were loaded onto sucrose gradients. AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers. Enzymatic activities are expressed in arbitrary units, and
representative sedimentation profiles are shown. (D) Sections from adult rat tibialis after 7 days of denervation (right) and sham-operated
(left) were triple stained with Alexa 647-conjugated a-bungarotoxin (pseudo-blue) for postsynaptic AChR, anti-synaptotagmin (SV48; red) for
presynaptic nerve terminal, and anti-PRiMA (green), and examined by confocal microscopy. Merged images allow a comparison of PRiMA
with presynaptic (SV48 + PRiMA) and postsynaptic (AChR + PRiMA) markers. The disappearance of the presynaptic nerve terminals in
denervated muscle is verified by the absence of SV48 labeling. PRiMA labeling was considerably reduced, but not completely absent. Representative images are shown, n = 3. Bar, 10 lm.

was dependent on the fast or slow nature of muscle
fibers. The strong expression of PRiMA protein and
G4 AChE in fast-twitch muscles is consistent with
previous results on PRiMA mRNA expression, i.e. the
tibialis contains an approximately 10-fold higher level
of PRiMA mRNA than the soleus [9]. The developmental change of PRiMA-linked G4 AChE in muscle
correlates with an increase in muscular activity and
muscle loading [15–17], which leads to the differentiation of fast-twitch and slow-twitch muscle fibers. The
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specific role of this AChE form at NMJs remains to be
elucidated.
Various forms of AChE exist in both developing
and mature NMJs. The major form is the asymmetric
collagen-tailed AChE, which is attached to the synaptic basal lamina [18]. Our study and others have shown
that G4 AChE is linked by PRiMA and localized in
the membranes of postsynaptic and presynaptic cells
[9]. At NMJs, three cell types can contribute to synaptic AChE: the postsynaptic muscle cell, the presynaptic

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motor neuron and the Schwann cell. During development, the muscle is the primary source of all forms of
AChE [1]. In contrast, the contribution of the Schwann cell, if any, is limited [14]; however, the possible
presence of PRiMA in the Schwann cell membrane
could only be distinguished by electron microscopy. In
this study, we confirmed the expression of PRiMA, as
well as of PRiMA-linked G4 AChE, in the motor neurons of the spinal cord using a PRiMA antibody. The
level of AChE increased during development, and was
reduced after section of the sciatic nerve. The current
results are in line with our previous observation that
chick motor neurons contain collagen-tailed AChE as
well as globular forms [15,19,20]. In contrast, frog
motor axons have been shown to produce collagentailed AChE, which could be deposited in the synaptic
basal lamina at NMJs [14]. The production of asymmetric AChE by motor neurons and its secretion by
the motor nerve terminals at frog NMJs could be
induced by damaged target muscles. Indeed, the capacity of a motor neuron to express asymmetric AChE at
an intact frog NMJ is still controversial.
In this study, confocal microscopy showed that
PRiMA-linked G4 AChE was found in both pre- and
postsynaptic membranes at NMJs. The distribution of
PRiMA appeared to be more extensive than that of
AChE. This may result from a higher sensitivity for
the detection of PRiMA. Alternatively, a fraction of
PRiMA may not be associated with AChET catalytic
subunits. For example, PRiMA can be associated with
butyrylcholinesterase (BChE). Indeed, the expression
of G4 BChE, together with G4 AChE, has been
revealed in brain and retina during development [21].

Our current and past results [15] indicate that motor
neurons represent the major cell type expressing
PRiMA and AChET in the spinal cord. In line with
this observation, it has been shown that AChE is
expressed in both neurotube and myotomes [22]. In
addition, previous studies have also shown that AChE
synthesized in the motor neuron is transported by axonal flow to the presynaptic terminal, as revealed by
enzymatic and microscopic studies [13]. The function
of pre- and postsynaptic PRiMA-linked G4 AChE
expressed by motor neuron and muscle, particularly
during early stages of development, is an open question. One of the proposed functions of two-sided
expression of AChE in both pre- and postsynaptic
membranes is to play an active role during synaptogenesis through the adhesive function of AChE [23,24].
In addition, the decrease in PRiMA and AChE expression in the rat spinal cord after section of the sciatic
nerve could be the consequence of trauma or of the
loss of retrograde influence from the muscle cells.

Proline-rich membrane anchor at neuromuscular junctions

Indeed, muscle-derived factors control the expression
of presynaptic proteins by motor neurons at NMJs
[17,25].
In previous studies, G4 AChE could only be identified by sucrose density gradients in the motor endplate
region [16,26]. In this study, we have provided the first
analysis of the expression of PRiMA at NMJs, using
an antibody specific for the cytoplasmic domain of
PRiMA I. In both fast-twitch and slow-twitch NMJs,
PRiMA was found in a peri-junctional region, suggesting that it is partly of muscle origin. Such a peri-junctional distribution of G4 AChE, which is more
abundant in fast-twitch than slow-twitch muscles [16],
may provide an AChE-rich environment embedding

NMJs and control the diffusion of acetylcholine out of
the synaptic cleft. However, most PRiMA-linked
G4 AChE was found to be located in the presynaptic
membrane of the motor nerve terminal. This is consistent with the presence of a significant amount of
AChE activity in the presynaptic membrane at NMJs
of the rat lumbricalis muscle [27]. The presence of
AChE in the presynaptic membrane can facilitate the
presynaptic re-uptake of choline resulting from the
hydrolysis of acetylcholine.

Materials and methods
Production of PRiMA antibody
The mouse PRiMA (amino acids 114–153)–glutathione
S-transferase fusion protein was expressed in BL21 (DE3)
pLysE Escherichia coli (Invitrogen, Carlsbad, CA, USA)
and purified by glutathione bead chromatography
(Amersham Biosciences, Piscataway, NJ, USA), according
to the manufacturer’s instructions. After digestion by
thrombin (Sigma, St Louis, MO, USA), the PRiMA
(amino acids 114–153) antigen was purified by Superdex
75 10 ⁄ 300 gel filtration chromatography (Amersham
Biosciences). Polyclonal antibodies were raised in a 2-kg
male New Zealand White rabbit by immunization with
750 lg of antigen, mixed with an equal volume of
complete Freund’s adjuvant (Sigma). The immunization
was carried out with the same amount of antigen three
times within 1 month. The anti-PRiMA serum was collected and purified by protein G-Sepharose (Amersham
Biosciences), according to the manufacturer’s instructions.
The amount of purified antibody was determined spectrophotometrically.

DNA construction and transfection
The HEK293T cell line was obtained from the American
Type Culture Collection (ATCC, Manassas, VA, USA) and

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Proline-rich membrane anchor at neuromuscular junctions

cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum. Cultured cells
were incubated at 37 °C in a water-saturated 5% CO2 incubator. All reagents for cell cultures were from Invitrogen.
cDNAs encoding full-length mouse PRiMA (PRiMA I) and
a COOH-terminal truncated mutant (PRiMADC-term;
obtained by deleting the COOH-terminal region, residues
122–153, of PRiMA I) were tagged with a FLAG epitope
(obtained by inserting the FLAG epitope DYKDE at position 36 between the putative signal sequence and the NH2
terminus) in pEF-BOS mammalian expression vector.
Transfection in cultured HEK293T was performed by
calcium phosphate precipitation.

Western blot analysis
HEK293T cultures or tissues were homogenized in lysis
buffer (10 mm HEPES, pH 7.5, 1 m NaCl, 1 mm EDTA,
1 mm EGTA, 0.5% Triton X-100 and 1 mgỈmL)1 bacitracin), followed by centrifugation at 12 000 g for 20 min at
4 °C. Protein samples were denatured at 100 °C for 5 min
in a buffer containing 1% SDS and 1% dithiothreitol,
and separated by 8% or 12% SDS–PAGE. For western

blot analysis, our PRiMA polyclonal antibody (purified at
0.5 lgỈmL)1), an AChE antibody E19 (1 : 2000; Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA), a monoclonal FLAG antibody (1 : 1000; Sigma) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies
(1 : 10 000; Sigma) were used. The immune complexes
were visualized using the enhanced chemiluminescence
method (Amersham Biosciences). The intensities of the
bands in the control and stimulated samples, run on the
same gel and under strictly standardized enhanced chemiluminescence conditions, were compared on an image
analyzer using, in each case, a calibration plot constructed
from a parallel gel with serial dilutions of one of the
samples.

Immunofluorescence analysis
Transfected cell cultures or tissue sections (16 lm) were
fixed by 4% paraformaldehyde in NaCl ⁄ Pi for 15 min,
followed by 50 mm ammonium chloride (NH4Cl) treatment
for 25 min. Samples were permeabilized by 0.2% Triton
X-100 in NaCl ⁄ Pi for 10 min and blocked by 5% BSA in
NaCl ⁄ Pi for 1 h at room temperature. Cultures were
stained with PRiMA (2 lgỈmL)1) or FLAG (1 : 500,
Sigma) antibodies. Tissue sections were double or triple
stained by rhodamine-conjugated or Alexa 647-conjugated
a-bungarotoxin (dilution 1 : 500; Molecular Probes,
Eugene, OR, USA), PRiMA antibody (2 lgỈmL)1), AChE
antibody (dilution 1 : 500, Santa Cruz Biotechnology), antisynaptotagmin (SV48) (1 : 500, BD Biosciences Clontech,
San Jose, CA, USA), anti-SNAP-25 (1 : 200, Sigma), antiChAT (1 : 200, Millipore, Bedford, MA, USA), anti-NeuN

3040

K. W. Leung et al.

(1 : 500, Millipore) and Cy3-conjugated anti-GFAP
(1 : 500, Sigma) for 16 h at 4 °C, followed by the corresponding fluorescence-conjugated secondary antibodies
(Alexa 488-conjugated anti-rabbit, Alexa 555- or Alexa
647-conjugated anti-mouse and anti-goat) for 2 h at room
temperature. The specificity of the PRiMA antibody was
established by pre-incubation with the PRiMA antigen
(10 lgỈmL)1) for 2 h at 4 °C. Samples were dehydrated
serially with 50%, 75%, 95% and 100% ethanol and
mounted with fluorescence mounting medium (DAKO,
Carpinteria, CA, USA). The samples were then examined
using a Leica confocal microscope with excitation at
488 nm ⁄ emission at 505–535 nm for green, excitation at
543 nm ⁄ emission at 560–620 nm for red, and excitation
at 647 nm ⁄ emission at 660–750 nm for pseudo-color.

Sucrose density gradient analyses
Separation of the various molecular forms of AChE was
performed by sucrose density gradient analysis, as
described previously [28]. In brief, sucrose gradients (5%
and 20%) in lysis buffer were prepared in 12 mL polyallomer ultracentrifugation tubes with a 0.4 mL cushion of
60% sucrose at the bottom. Sample extracts (0.2 mL)
mixed with sedimentation markers (alkaline phosphatase,
6.1S; b-galactosidase, 16S) were loaded onto the gradients
and centrifuged at 175 000 g in a Sorvall TH 641 rotor at
4 °C for 16 h. Approximately 45 fractions were collected
and AChE enzymatic activity was determined according to
the method of Ellman [29]; the reaction medium contained
0.1 mm tetra-isopropylpyrophosphoramide, an inhibitor of
BChE. Absorbance at 410 nm was recorded as a function

of the reaction time. The proportions of the various
AChE forms were determined by summation of the enzymatic activities corresponding to the peaks of the sedimentation profile. In the immunoprecipitation of G4 AChE by
PRiMA antibody, 1 mL samples of tissue extracts were
incubated for 4 h at 4 °C with purified PRiMA antibody
(10 lgỈmL)1). Then, 50 lL of washed protein-G agarose
gel (Santa Cruz Biotechnology) was added and incubated
for 1 h at 4 °C. After centrifugation, the supernatants
were loaded onto sucrose gradients for sedimentation
analysis.

ELISA for PRiMA-linked G4 AChE
Fifty microliter samples of serially diluted PRiMA antibody
were coated in a 96-well ELISA plate (Nunc Maxisorp
Immunoplate, Roskilde, Denmark) for 16 h. The antibody
was removed and the plate was washed twice with 200 lL
NaCl ⁄ Pi containing 0.1% Tween-20. The plate was blocked
by NaCl ⁄ Pi with 5% fetal bovine serum for 2 h at room
temperature. Tissue lysates containing equal AChE activity
were loaded onto the precoated ELISA plate and incubated
for 2 h. The plate was washed three times with 200 lL

FEBS Journal 276 (2009) 3031–3042 ª 2009 The Authors Journal compilation ª 2009 FEBS


K. W. Leung et al.

Proline-rich membrane anchor at neuromuscular junctions

NaCl ⁄ Pi containing 0.1% Tween-20, and the retained
AChE activity was measured.

significant for P < 0.05 and P < 0.01 and highly significant for P < 0.001.

Real-time PCR analysis

Acknowledgements

Total RNA from rat tissues was isolated with TRIzol
reagent (Invitrogen), and 5 lg of RNA was reverse transcribed by Moloney Murine Leukemia Virus Reverse
Transcriptase (Invitrogen), according to the manufacturer’s
instructions. Real-time PCR of PRiMA, AChET and 18S
transcripts was performed on equal amounts of reversetranscribed products, using SYBR Green Master mix and
Rox reference dye, according to the manufacturer’s instructions (Applied Bioscience, Foster City, CA, USA). The
primers were as follows: 5¢-TCTGACTGTCCTGGTCATC
ATTTGCTAC-3¢ and 5¢-TCACACCACCGCAGCGTT
CAC-3¢ for mouse PRiMA I and II (GenBank numbers
NM 133364 and NM 178023); 5¢-CTGGGGTGCGGA
TCGGTGTACCCC-3¢ and 5¢-TCACAGGTCTGAGCAG
CGTTCCTG-3¢ for mouse AChET [30]; 5¢-TGTGATGC
CCTTAGATGTCC-3¢ and 5¢-GATAGTCAAGTTCGAC
CGTC-3¢ for rat 18S ribosomal RNA. The SYBR green
signal was detected by an Mx3000pÔ multiplex quantitative
PCR machine (Stratagene, La Jolla, CA, USA). Transcript
expression levels were quantified using the DDCt value
method [31], where values were normalized to 18S rRNA
as an internal control in the same sample. PCR products
were analyzed by gel electrophoresis and the specificity of
amplification was confirmed by the melting curves.

This work was supported by the Research Grants

Council of Hong Kong (HKUST 6404 ⁄ 05M,
6419 ⁄ 06M, 662407, 662608) to KWKT.

Sciatic nerve section
Two-month-old Sprague–Dawley rats weighing approximately 250 g were anesthetized by isoflurane. A portion of
approximately 3 mm of the sciatic nerve located around
the upper thigh was removed by an aseptic surgical technique [13]. The rats were sacrificed according to the
instructions of the Animal Care Facility of The Hong
Kong University of Science and Technology. Spinal cord
(lumbar) and tibialis muscles were collected 7 days after
denervation. Samples were frozen in liquid nitrogen immediately after dissection and stored at )80 °C for RNA and
protein extraction, and for confocal microscopy. Control
experiments were performed by sham operation on different rats.

Other assays
Protein concentrations were measured by Bradford’s
method [32] with a kit from Bio-Rad Laboratories (Hercules, CA, USA). Statistical tests were performed by the
primer program, version 1 [33]: differences from basal or
control values (as shown in the plots) were classified as

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