(Received for publication, July 19, 1996, and in revised form, November 15, 1996)
From the Neuropeptide Y (NPY) is a 36-residue-long
neuropeptide which has been implicated in the regulation of feeding
behavior and modulation of the circadian rhythm. We identified the
primary structure of the endogenous NPY-immunoreactive material in the rat hippocampus using a combination of chromatographic techniques and
nanospray mass spectrometry. The major component in the brain tissue
corresponded to the authentic amidated form of NPY(1-36). The fate of
NPY in the central nervous system was studied by subjecting pure
peptide to the protease(s) present in hippocampal synaptosomes to
reveal potential cleavage site(s). NPY was efficiently metabolized with
a single cleavage between Leu30-Ile31. Thus,
processing of NPY resulted in formation of the C-terminally truncated
fragment NPY(1-30) and its counterpart NPY(31-36). The enzyme
revealed properties of aspartic protease, being blocked by pepstatin
and having a pH optimum between 4 and 5. The data clarify the structure
of NPY and its inactivation pathway in the brain, which is different
from that found in the periphery, and may have important consequences
in vivo.
Neuropeptide Y (NPY)1 is a
36-amino-acid-long peptide with an amidated C terminus, first isolated
by Tatemoto and co-workers in 1982. The peptide belongs to the
pancreatic polypeptide family (1) and is one of the most abundant found
in the central nervous system (2). NPY has been implicated in several
central regulatory functions such as circadian rhythm (3) and feeding
behavior (4). Stimulation of either the central noradrenergic or NPY pathways activates the hypothalamic-pituitary-adrenocortical axis in
the rat and in conscious sheep (5). In a recent study (6) we found
increased concentrations of NPY-like immunoreactivity (NPY-LI) in
specific rat brain regions following repeated electroconvulsive shocks.
The measured NPY-LI consisted of intact NPY(1-36) and a component
anticipated to be NPY sulfoxide. This finding was thought to be of
importance due to different biological properties of NPY(1-36) as
compared to the effects of its C-terminal homologues (7, 8) on the
different receptor types for NPY (8-11). To characterize the existing
peptide pool in a tissue, various chromatographic techniques combined
with immunochemical methods are usually used. However, one limiting
factor in the identification of the peptide homologues in biological
material is the specificity of the antiserum used.
Little is known about the fate of NPY once it is released into the
synaptic cleft in the central nervous system. The C terminus of NPY is
amidated, protecting the molecule against an attack of
carboxypeptidases. So far, it has only been shown that NPY is degraded
in the periphery by endopeptidase-2 (12) and dipeptidyl peptidase IV
(13). In a study on cultivated neurons and glial cells (14) it has been
found that serine protease can play an important role in extracellular
processing of NPY. In the present study we intended to reveal the
structure of existing endogenous NPY in the rat brain and to
investigate its inactivation using nanospray mass spectrometry
(ESI-MS). This technique, based on the very low flow rates, utilizes
extremely low amounts of biological material which can be unambiguously
characterized for a relatively long time (15). Hippocampus was chosen
in this study because it contains high concentrations of NPY and
because this tissue is the major site for the Y2 receptor, which has a
high affinity to C-terminally truncated homologues of the peptide (16,
17).
Synthetic human/rat NPY was purchased from
Peninsula (St. Helens, United Kingdom). The C1-NPY antiserum was
obtained from professor Elvar Theodorsson, University Hospital,
Linköping, Sweden. Proteinase inhibitors and 1-heptanesulfonic
acid were purchased from Sigma-Aldrich (Stockholm,
Sweden), and CoCl2, ZnCl2, HgCl2,
and CaCl2 were from ICN Pharmaceuticals (Costa Mesa, CA). Laboratory reagents of the analytical grade were purchased from Merck
unless indicated otherwise.
Ten
male Sprague-Dawley rats (ALAB, Sollentuna, Sweden) weighing 250 g
were sacrificed by decapitation. Immediately after death, the
hippocampi were dissected according to the method of Glowinski and
Iversen (18), weighed, and stored at NPY-LI was analyzed using the C1 antiserum
which cross-reacts with human/rat NPY (100%), porcine NPY (107%),
sulfoxidated NPY (88%), peptide YY (63%), including their C-terminal
fragments, except (Leu31-Pro34)-NPY and
NPY(1-21) (6). HPLC-purified
125I-Bolton-Hunter-labeled NPY was used as radioligand and
human/rat NPY as a standard.
The sample was redissolved in
2.5 ml of H2O and passed through a 0.45-µm Millipore
filter before being applied onto a Sephadex G-50 Superfine column
(2.5 × 95 cm, Pharmacia Biotech Inc.). The column was eluted with
720 ml of 1 M acetic acid at a flow rate of 0.5 ml/min.
Four-ml fractions were collected, and 10-µl aliquots of each fraction
were tested for NPY-LI with a competitive radioimmunoassay. The column
was calibrated in a separate run with 1 pmol of synthetic human/rat
NPY(1-36).
Two different columns were subsequently used for HPLC
purifications. A Delta Pak RP-HPLC column (3.9 × 150 mm, Waters
Sverige AB, Sweden) was connected to the SMART micropurification system (Pharmacia) and eluted with a 40-ml linear gradient of acetonitrile (20-50%) supplemented with 0.1% trifluoroacetic acid. Flow rate was
maintained at 0.5 ml/min. One-ml fractions were collected and 10-µl
aliquots of each fraction were tested for NPY-LI. Active fractions were
pooled and applied on a µRPC C2/C18 SC 2.1/10 RP-HPLC column
(2.1 × 100 mm, Pharmacia) attached to the SMART system. The
column was eluted with a 5-ml linear gradient of acetonitrile (32-43%) supplemented with 0.1% trifluoroacetic acid. Flow rate was
adjusted to 0.1 ml/min. 100-µl fractions were collected and the
NPY-LI was assessed in 5 µl of each fraction, as described above.
A PepRPC HR 5/5 column (5 × 50 mm, Pharmacia) connected to the SMART system via a short column
holder (Pharmacia) was used. Immunoreactive fractions collected from
the µRPC column and corresponding to the intact NPY were pooled, and
a part of it was subjected to the ion-pair chromatography. The column
was eluted with a 40-ml linear gradient of methanol (20-100%)
supplemented with 0.1% trifluoroacetic acid and 0.1% heptanesulfonic
acid at a flow rate of 1 ml/min. 0.5-ml fractions were collected and
the NPY-LI content was measured in 5-µl aliquots of each
fraction.
The system used
was a Finnigan MAT 95Q (Finnigan MAT, Bremen, Germany) equipped with an
atmospheric pressure ionization source, as described previously (19).
Immunoreactive samples containing endogenous NPY material were
introduced via a Valco injector equipped with a 5-µl loop. The loop
was loaded with the analyte and the material was eluted at a flow rate
of 200 nl/min. No sheath gas was applied in this case.
Liquid chromatography-mass spectrometry was performed essentially as
described in a previous paper (19) using the SMART system connected to
the ESI interface. The samples were separated on a reversed-phase
column µRPC C2/C18 (2.1 × 100 mm) using a 3-ml linear gradient
(0-60%) of acetonitrile/0.1% trifluoroacetic acid. The flow rate was
maintained at 0.1 ml/min.
The NPY sample was
desalted on a fast desalting column (Pharmacia). After evaporating to
near dryness, the sample was digested overnight with 0.5 µg of
modified trypsin (Promega, Madison, WI) in 0.1 M ammonium
bicarbonate at 30 °C. The generated peptides were isolated by
RP-HPLC on a µRPC C2/C18 SC prototype column (1 × 100 mm,
Pharmacia) connected to the SMART system, using a 4-ml
acetonitrile/0.05% trifluoroacetic acid gradient (0-40%) at a flow
rate of 25 µl/min. By comparing with the SwissProt (updated 8/18/96)
and NCBI (version 9/5/96) data bases available on the Internet, the
unique pattern of tryptic fragments detected by ESI-MS was identified
(20). The searching programs were PeptideSearch (EMBL Protein and
Peptide Group, Heidelberg, Germany) and the MS-Fit (UCSF Mass
Spectrometry Facility, San Francisco, CA). The monoisotopic mass
tolerance was set to 0.5 Da, and the number of missed cleavages was set
to 1. The charge states of the particular ions were determined using
isotope distribution measured at a moderate resolution of 3500 (10%
valley) and by applying PepMatch software supplied by the Finnigan
MAT.
Synaptosomes were
isolated from four hippocampi following a procedure described elsewhere
(21). The synaptosomal pellet was resuspended in 500 µl of 10 mM Tris-HCl buffer (pH 7.4) and kept at The
extracted sample from rat hippocampi was submitted to size-exclusion
chromatography on a Sephadex G-50 Superfine column and eluted as two
major components (fractions 71-78 and 145-150) and one minor
component (fractions 104-112) of NPY-LI (Fig.
1A). The first component co-eluted with
synthetic human/rat NPY(1-36). Further purification of the NPY-LI
fractions was achieved by two consecutive RP-HPLC runs. In the first
step, fractions 71-78 and 145-150 from the size-exclusion
chromatography were pooled and applied on the HPLC column in separate
runs; however, only the first pool possessed NPY-LI (Fig.
1B). Fractions 16-19, co-eluting with authentic NPY, from
the first RP-HPLC run were pooled and subjected to the second RP-HPLC
purification (Fig. 1C). The NPY-LI material present in
fractions 13-14 from the second RP-HPLC step were pooled, evaporated,
and then redissolved in 25 µl of 30% acetonitrile/0.1% formic acid.
This material was directly analyzed (two times at 400 fmol) by
nanospray mass spectrometry. Fig. 2A shows
the obtained mass spectrum containing the multiply charged species of
rat NPY at m/z of 610.1 (+7), 712.9 (+6), and 855.7 (+5).
The spectra were accumulated and deconvoluted as shown in Fig.
2B, revealing the molecular mass of 4270 corresponding to the intact sequence of rat/human NPY(1-36). The content of fractions 8-9 (Fig. 1C) was analyzed in a similar way by the ESI-MS.
This resulted in detection of the salt adducts (data not shown), and no
peptide could be seen in this sample. The purity of the preparation subjected to nanospray analysis was estimated using intensities of the
contaminating ions shown in Fig. 2A (integrated area of each
component in the ion chromatogram). The molecular masses of the
contaminant(s) did not correspond to any pro-NPY-derived fragment. The
NPY preparation was approximately 80% homogenous; therefore it was
further purified by ion-pair RP-HPLC (Fig. 1D) before
tryptic digestion. The NPY structure was verified by searching the
protein data bases (SwissProt and NCBI) for the unique set of tryptic
fragments prepared from the additional 4 pmol of endogenous material
(Fig. 1D). Three fractions were identified along the chromatogram: fragment T2 at m/z 772.4 (NPY 20-25); T3 at
m/z 1029.6 (NPY 26-33), and a triply protonated ion T1+T2
at m/z 938 (NPY 1-25) (molecular mass 2812.3). The
MassSearch program does not list the number of entries searched. In
contrast, the MS-Fit program has selected 5,500 of 209,493 entries,
using a combination of molecular weight (0.1-100 kDa) and defined
species (Rattus norvegicus). The results from both searching
procedures suggested only one solution to this combination and thus was
consistent with the intact NPY molecule (SwissProt, P09640; NCBI,
100226 3 128120).
Metabolism of NPY in
the rat hippocampus was studied using both membrane preparations
(synaptosomal) and cytosolic extracts at various pH values. An on-line
liquid chromatography-mass spectrometry experiment was performed to
disclose any suppressed fragments which could be missed during direct
analysis (Fig. 3A). Human/rat NPY was
efficiently cleaved by the proteinase present in the synaptosomal fraction of the hippocampus. ESI-MS revealed that NPY was processed between Leu30-Ile31. The cleavage gave rise to
two fragments, NPY(1-30), with a molecular mass of 3454, and its
C-terminal counterpart NPY(31-36), with a molecular mass of 835 (Fig.
3B).
The proteolytic activity cleaving NPY was studied using various
proteinase inhibitors. Table I summarizes the data,
which indicate that pepstatin, Zn2+, and Hg2+
were the most effective. Time-dependent studies using
pepstatin showed that this inhibitor was effective even during a
prolonged incubation time (data not shown). The pH optimum for the
proteolytic enzyme was between 4 and 5 (Fig. 4).
Influence of various agents on NPY metabolism
This study, based on the mass spectrometric verification of the
peptide identity, indicates the presence of an intact NPY(1-36) molecule in the rat brain hippocampus. Our earlier findings, based on
an immunological technique, anticipated that only
NPY(1-36)-immunoreactive material could be detected in this tissue
(6), which consisted of NPY and its sulfoxidated form. The limiting
factor in such studies is the specificity of the antiserum used which,
in our study, was directed toward the C-terminal portion of the NPY
molecule (6). Sulfoxidated NPY (if existing) should have been eluted around fractions 8-9 (Fig. 1C) from the reversed-phase
column under the conditions tested. The estimated amount of material present in this immunoreactive pool is low (approximately 25%) in
relation to the total NPY-LI content, and this observation is
consistent with our previous experiments (6). A relatively low amount
of material and high salt content could simply mask the presence of
another peptidergic component. The low molecular weight component
(fractions 145-150, Fig. 1A) was subjected to further
separation on the RP-HPLC column. Apparently, this immunoreactive pool
lost its reactivity against the NPY antiserum after further purification. One explanation could be that the fractions are eluted
near the total volume of the column (fractions 148-155) and
that low molecular weight contaminants may contribute to the false-positive NPY-like immunoreactivity.
Using the nanospray MS technique, we were able to verify that the
fractions showing NPY-LI and co-eluting with synthetic peptide in the
last chromatographic step mainly consisted of a component having a
molecular mass of 4270, corresponding to the intact sequence of
amidated rat/human NPY(1-36). This also indirectly confirms the
specificity of the antiserum used in this study. Furthermore, the
identity of the endogenous NPY was verified using a set of peptide
fragments produced after limited digestion with trypsin. Mass
spectrometric peptide mapping combined with protein data base search is
a very efficient technique for the rapid identification of isolated
peptides and proteins (22, 23). Here, we applied this strategy to
confirm the identity of NPY(1-36) in the rat hippocampus.
Several proteinases cleaving NPY have been described, including
aminopeptidase P, dipeptidylpeptidase IV (13, 24), and a
phosphoramidon-insensitive endopeptidase (endopeptidase-2) (12). All of these enzymes originated from the peripheral systems. The metabolic pathway of NPY in the central nervous system, however, is yet
unknown. In a recent study, Ludwig and co-workers (14) showed that
cultivated neurons and microglia digested NPY with a cleavage pattern
resembling the one observed when NPY was degraded by purified
urokinase-type plasminogen activator, plasmin, thrombin, and trypsin.
Thus, it was suggested that serine proteinases might play an important
role in extracellular neuropeptide processing under physiological
conditions. The present study shows that human/rat NPY was efficiently
cleaved at an acidic pH by the proteinase present in the synaptosomal
fraction of rat hippocampus. The cleavage pattern revealed that NPY is
processed between Leu30-Ile31, thus giving rise
to two fragments, NPY(1-30) at molecular mass 3454, and its C-terminal
counterpart NPY(31-36), at molecular mass 835. The proteolytic
activity was completely blocked by 1 µM pepstatin, thus
classifying the enzyme as belonging to the family of aspartic
proteinases. The enzyme was also partially blocked by the presence of
Zn2+ and Hg2+. An intact C-terminal end of the
NPY molecule is essential for its action upon the Y1 and Y2 receptors.
For a full recognition by the NPY Y1 receptor, both the C- and
N-terminal parts of the molecule are necessary. Elongated C-terminal
homologues are more potent on the Y2 receptor than shorter ones (25).
Thus, the C-terminal truncation of NPY observed in the present study
would totally hamper the action of NPY. The cleavage pattern causing formation of NPY(1-30) and NPY(31-36) described in this work is new
and cannot be caused by the presently known peptidases.
Dipeptidylpeptidase IV liberates N-terminal dipeptide Tyr-Pro (13, 24),
and the specificity of endopeptidase-2 was tested using a radioactive substrate without structural identification of the fragments formed during the enzymatic process (26). Nevertheless, this latter enzyme is
optimally active at neutral pH and was efficiently inhibited by
chelating agents, whereas the enzyme described in this paper was active
at acidic pH and was classified as an aspartic protease. The
physiological function of the described enzyme is not known. One
possible explanation is that NPY is internalized into the neuron after
it has been bound to its receptor, followed by its termination by
vesicular enzymes. In this context it is interesting to mention the
recent work on neurotensin (27), demonstrating the existence of a
retrograde axonal transport for neurotensin in the rat brain. This
peptide can be retrogradely transported in dopaminergic nigrostriatal
neurons, and such transport first involves binding of neurotensin to
its receptors presynaptically located on dopamine nerve terminals of
the nigrostriatal pathway. Then the ligand-receptor complex can be
rapidly internalized. Finally, the complex enters the perikaryon and is
processed along a variety of intracellular pathways, including
lysosomal inactivation.
In the present study we applied various liquid chromatographic
techniques in combination with electrospray ionization mass spectrometry. This strategy was used for characterization of endogenous NPY in central nervous system tissue and also for a rapid and unambiguous verification of the cleavage pattern of the neuropeptide in
selected brain structures. The detailed knowledge on NPY metabolism in
the central nervous system may lead to the design and synthesis of
specific inhibitors having potential therapeutic applications in,
e.g., problems with food intake and metabolism, which are at
least partially dependent on NPY biosynthesis and release (17, 28,
29).
We thank professor Elvar Theodorsson for the
kind gift of NPY antiserum. We thank Anja Finn for experienced
technical assistance.
Department of Laboratory Medicine and
Department of Clinical Neuroscience,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
Materials
80 °C until extraction. The
frozen hippocampi (approximately 1.2 g) were cut into small pieces
and boiled for 10 min in 10 volumes of 1 M acetic acid.
After homogenization, the sample was centrifuged at 2500 × g for 10 min. The supernatant was lyophilized and stored at
80 °C until used.
80 °C until
used. The synaptosomal preparation was sonicated before use. The
typical assay mixture consisted of 10 µl of the synaptosomal
preparation (diluted 20 times with the appropriate buffer) and 5 µg
of human/rat NPY as a substrate in a total volume of 20 µl. Aliquots
were withdrawn after various time intervals, adjusted to 10 µl with
30% acetonitrile/0.1% formic acid, and subjected to analysis by
ESI-MS. Inhibitory studies were performed under the above described
conditions at optimal pH (pH 4), but the enzyme preparations were
preincubated with the respective inhibitor for 30 min at 37 °C
followed by addition of the substrate. Appropriate blanks were added
where necessary.
Characterization of Endogenous NPY in Rat Hippocampus
Fig. 1.
Chromatographic profile of NPY-like
immunoreactive material extracted from 10 rat hippocampi. Material
was extracted after size-exclusion chromatography on a Sephadex G-50
column (2.5 × 95 cm) (A), RP-HPLC on a Delta Pak
column (3.9 × 150 mm) eluted with a gradient of 20-50%
acetonitrile/trifluoroacetic acid (B), RP-HPLC on a µRPC
C2/C18 SC 2.1/10 (2.1 × 100 mm) eluted with a gradient of
32-43% acetonitrile/trifluoroacetic acid (C), and ion-pair
RP-HPLC on a PepRPC HR 5/5 (5 × 50 mm) column eluted with a
gradient of 20-100% methanol/trifluoroacetic acid/heptanesulfonic acid (D). Fractions were collected and NPY-LI analyzed by
radioimmunoassay. The dotted lines indicate the gradients
used. The elution position of synthetic NPY(1-36) is indicated with an
arrow in A.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Characterization of endogenous NPY by
nanospray MS. A, accumulated mass spectrum showing the
multiply charged species of endogenous NPY. m/z,
mass-to-charge ratio. B, deconvoluted ESI spectrum revealing
the molecular weight of intact amidated NPY.
[View Larger Version of this Image (14K GIF file)]
Fig. 3.
Degradation of synthetic human/rat NPY by the
enzyme present in rat hippocampal synaptosomes. A, liquid
chromatography-mass spectrometry RP-HPLC analysis of the fragments
formed during proteolysis. Mass chromatogram shows multiply charged
ions corresponding to the particular NPY fragments NPY(1-36),
NPY(1-30), and NPY(31-36) and their retention times. B,
partial ESI spectrum showing the NPY fragments (multiply-charged ions)
detected during liquid chromatography-mass spectrometry analysis.
[View Larger Version of this Image (18K GIF file)]
Peptidase
inhibitor
Concentration
Enzyme activity
%
control
No inhibitor
100
EDTA
1 mM
50
Phenylmethylsulfonyl
fluoride
1 mM
50
EDTA + phenylmethylsulfonyl
fluoride
1 mM + 1 mM
65
p-Hydroxymercaribenzoic
acid
0.25 mM
100
Captopril
10 µM
100
Phosphoramidon
100 µM
100
CoCl2
1 mM
100
ZnCl2
1 mM
10
HgCl2
1 mM
2
CaCl2
1 mM
100
Pepstatin
1 µM
0
Fig. 4.
pH dependence of the proteolytic activity
cleaving human/rat NPY in rat hippocampal synaptosomes. The
synaptosomes were diluted 20 times with buffers with different pH.
Formation of NPY(1-30) was used as a measure of the proteolytic
conversion (quantified by ESI-MS) and related to the amount of peptide
released at optimal pH (4.0).
[View Larger Version of this Image (15K GIF file)]
*
This work was supported by the Wenner-Gren Center
Foundation, the Swedish Society of Medicine, The Thuring Foundation,
The Golje Memory Foundation, and Karolinska Institute Research Funds. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Behavioural and
Biochemical Pharmacology, Astra Arcus AB, S-151 85 Södertälje, Sweden. Tel.: 46-8-55326362; Fax:
46-8-55328891; E-mail: carina.stenfors{at}arcus.se.astra.com.
**
Supported by the Swedish Alcohol Research Fund, the Swedish Medical
Research Council, and KBN Grant 1147/IA/158/95.
1
The abbreviations used are: NPY, neuropeptide Y;
NPY-LI, NPY-like immunoreactivity; ESI-MS, nanospray mass spectrometry; RP-HPLC, reversed-phase high performance liquid chromatography.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.