From the Department of Pharmacology, New York University School of
Medicine, New York, New York 10016 and the Department
of Molecular Pharmacology, Albert Einstein College of Medicine,
Bronx, New York 10461
Received for publication, October 4, 2000, and in revised form, November 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ProSAAS is a newly discovered protein with a
neuroendocrine distribution generally similar to that of prohormone
convertase 1 (PC1), a peptide-processing endopeptidase. Several
proSAAS-derived peptides were previously identified in the brain and
pituitary of the Cpefat/Cpefat
mouse based on the accumulation of C-terminally extended peptides due
to the absence of enzymatically active carboxypeptidase E, a
peptide-processing exopeptidase. In the present study, antisera against
different regions of proSAAS were used to develop radioimmunoassays and
examine the processing profile of proSAAS in wild type and Cpefat/Cpefat mouse tissues
following gel filtration and reverse phase high performance liquid
chromatography. In wild type mouse brain and pituitary, the majority of
proSAAS is processed into smaller peptides. These proSAAS-derived
peptides elute from the reverse-phase column in the same positions as
synthetic peptides that correspond to little SAAS, PEN, and big LEN.
Mass spectrometry revealed the presence of peptides with the expected
molecular masses of little SAAS and big LEN in the fractions containing
immunoreactive peptides. The processing of proSAAS is slightly impaired
in Cpefat/Cpefat mice, relative
to wild-type mice, leading to the accumulation of partially processed
peptides. One of these peptides, the C-terminally extended form of PEN,
is known to inhibit PC1 activity and this could account for the
reduction in enzymatically active PC1 seen in
Cpefat/Cpefat mice. The
observation that little SAAS and big LEN are the major forms of these
peptides produced in mouse brain and pituitary raises the possibility
that these peptides function as neurotransmitters or hormones.
The majority of peptide hormones and peptide neurotransmitters are
synthesized as larger precursors that undergo limited endoproteolysis at specific sites (1, 2). Initially, the precursor is cleaved by
endoproteases at basic residues. This is followed by carboxypeptidases that remove the C-terminal basic residue extensions from peptides (3).
In some cases, peptides with C-terminally extended Gly residues are
further processed into C-terminally amidated peptides by
peptidyl-glycine- Endoproteases of the subtilisin family of serine proteases cleave
peptide precursors at basic residue cleavage sites (5-7). These
enzymes are homologous to KEX-2, a yeast peptide-processing enzyme, and
have been designated prohormone convertases
(PCs)1 (5-8). Among the
members of the PC family, PC1 (also known as PC3) and PC2 exhibit a
restricted neuroendocrine distribution, suggesting that these enzymes
play a key role in the endoproteolytic processing of a number of
neuropeptides and peptide hormones (9-11).
Carboxypeptidase E (CPE; also known as EC 3.4.17.10 and CPH) is the
enzyme responsible for the C-terminal trimming of the majority of
peptide hormones and neurotransmitters (3). Support for the involvement
of CPE in the processing of neuroendocrine peptides comes from studies
on the Cpefat/Cpefat mouse. These
mice lack active CPE due to a point mutation (serine 202 to proline) in
a highly conserved region of the enzyme (12). Without CPE activity,
these mice show deficiency in C-terminal trimming of basic residues
from the processing intermediates (13, 14). In addition, these mice
exhibit impaired endoproteolytic processing as characterized by
precursor accumulation (12-15). These changes point to an alteration
in the endoproteolytic processing of neuropeptides and hormone
precursors in Cpefat/Cpefat mice.
Using affinity chromatography to isolate substrates of CPE from the
Cpefat/Cpefat mice
(i.e. Lys- and Arg-extended peptides), a number of novel peptides were identified (16). Molecular cloning revealed that several
of these peptides belonged to a single precursor, designated "proSAAS" (16). ProSAAS mRNA is expressed primarily in the
brain and other neuroendocrine tissues. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at
paired basic cleavage sites into smaller peptides. ProSAAS was found to
be a potent inhibitor of PC1 in vitro (16), and the
inhibitory region was mapped to a short 8-residue sequence near the C
terminus of the protein (17, 18). ProSAAS showed some similar
characteristics to granins, a loosely defined family of neuroendocrine
proteins that includes chromogranin A, chromogranin B, secretogranin
II, and a protein designated 7B2. These granin proteins have been shown
to undergo proteolytic processing in neuroendocrine tissues (19-21).
However, it is not clear if the primary function of these proteins is
as neuropeptide precursors.
To gain insight as to the function of proSAAS and the proSAAS-derived
peptides, it is important to identify the major forms of this protein
in wild-type mouse brain and pituitary. The finding that the majority
of proSAAS is processed into smaller peptides raises the possibility
that these peptides possess physiological functions, possibly as
neuroendocrine hormones or neurotransmitters.
Characterization of the Antisera Using Radioimmunoassays
(RIAs)--
The antisera to proSAAS-(42-59) (little SAAS),
proSAAS-(221-242) (PEN), and proSAAS-(245-260) (big LEN) were the
generous gift of Dr. James Douglass (Amgen, Thousand Oaks, CA). These
antisera were raised against synthetic peptides with additional
N-terminal Cys residues that were conjugated to maleimide-activated
keyhole limpet hemocyanin. Each of the three peptides was used for
production of antiserum in three rabbits. These nine antisera were
screened against radioiodinated peptides with amino-terminal Tyr
synthesized for this purpose; peptides were iodinated by the
Chloramine-T method, and the radiolabeled tracers were purified on a
PD-10 gel filtration column (Sephadex G-25M; Amersham Pharmacia
Biotech). Of the three antisera to each peptide, the one with the
highest sensitivity was selected for further studies: little SAAS
(rabbit number 2766), PEN (rabbit number 141), and big LEN (rabbit
number 85). Synthetic peptide standards were prepared in
phosphate-buffered saline. Antibody dilutions that bind 30% of the
radioactivity (1:3000 for anti-SAAS and anti-LEN and 1:4500 for
anti-PEN) were added in a RIA buffer (150 mM sodium
phosphate buffer, pH 7.5, containing 0.1% gelatin (Bio-Rad), 0.1%
bovine serum albumin (protease-free; Sigma), 0.1% Triton X-100, and
0.02% sodium azide); among the buffers tested, this RIA buffer was
found to decrease nonspecific binding and increase the sensitivity of
the assay. The tubes were incubated overnight at 4 °C, and then 100 µl of goat anti-rabbit globulin and 100 µl of normal rabbit serum
were added. The antigen-antibody complex was separated from the unbound
radioligand according to the manufacturer's protocol (Peninsula).
The concentrations of nonradioactive peptides required to reduce tracer
binding by 50% (i.e. IC50 values) were 900 fmol/tube for SAAS, 300 fmol/tube for PEN, and 200 fmol/tube for LEN,
and the IC20 values were 200 fmol/tube for SAAS, 90 fmol/tube for PEN, and 25 fmol/tube for LEN. To determine the
specificity of each antiserum, synthetic peptides or glutathione
S-transferase fusion proteins representing various portions
of proSAAS (17) were used (see Fig. 1 for the position of these
peptides within the proSAAS sequence).
Tissue Preparation--
Balb/c mice (Taconic Farms, NY),
Cpefat/Cpefat C57BLKS/J mice and
wild type littermates (Jackson Laboratory, Bar Harbor, ME) were maintained in individual cages on a 12-h light/dark cycle. The animals
were sacrificed by decapitation, and the brains were removed. Gross
dissection of brain regions was according to Glowinski and Iversen
(22). The pituitary was separated into anterior (AL) and
neurointermediate (NIL) lobe under a dissecting microscope. Dissected
tissues were kept at Peptide Extraction--
Tissues were extracted in 10 volumes of
one of five different buffers (described below), homogenates were
subjected to centrifugation (13,000 × g for 30 min,
4 °C), and the pellets were reextracted with 10 volumes of second
extraction buffer (see below). Following recentrifugation, supernatants
from both extractions were dried separately using a Speed Vac
concentrator (Savant) and stored at
To determine the optimal extraction conditions, five different
protocols were tested: (1) homogenization in boiling water followed by
incubation at 100 °C for 10 min and a second extraction in ice-cold
1 M acetic acid, (2) homogenization in boiling 0.15 M acetic acid followed by incubation at 100 °C for 10 min and a second extraction in 0.1 M acetic acid/methanol
(1:1), (3) homogenization in ice-cold 1 M acetic acid and a
second extraction in methanol plus 0.1 M acetic acid
(1:1), (4) homogenization in ice-cold ethanol plus 0.8 N
HCl (3:1) with no second extraction, and (5) homogenization in ice-cold
Tris buffer plus 1 M acetic acid (1:1) and a second extraction in ethanol plus 1 M acetic acid (1:4); the Tris
buffer consisted of 50 mM Tris-Cl, pH 7.5, containing 0.2%
Triton X-100, 280 µM tosyl-prolyl chloromethyl ketone,
140 µM tosyl-lysyl chloromethyl ketone, 1 µM E-64, and 1 µM pepstatin. Among these,
the first extraction condition was found to be optimal, since the level of immunoreactive peptides were ~2-fold higher under these conditions as compared with other conditions. Also, the recovery of radiolabeled peptides using the first extraction condition was 85-90%, indicating no significant breakdown of peptides during extraction. Furthermore, when the material from the first and second extraction conditions was subjected to gel filtration chromatography (described below) comparable profiles were seen for all three peptides examined. Therefore, the first extraction conditions were routinely used for
further characterization.
Size Exclusion Chromatography and RIAs--
Gel exclusion
chromatography was performed on Superdex Peptide HR 10/30 column
(Amersham Pharmacia Biotech). The sample in a total volume of 50-100
µl was applied to the column and separated with 0.1% trifluoroacetic
acid containing 30% acetonitrile (Fisher). The flow rate was 0.5 ml/min; 0.5-ml fractions were collected. Fractions were dried and
resuspended in sodium phosphate buffer, pH 7.5, containing 0.2% Triton
X-100 and subjected to RIA.
Reverse-phase High Performance Liquid Chromatography (HPLC)
Separation of the Immunoreactive Material in the 2-3-kDa
Peak--
The material representing the highest immunoreactive
peptides was pooled and subjected to HPLC on a 5-µm, 250 × 4.6-mm C18 column (Column Engineering, Inc., Ontario, CA).
A linear gradient of 0-75% acetonitrile in 0.1% trifluoroacetic acid
was used to separate the peptides (75 min, 1 ml/min, 0.5-min
fractions). Retention times of synthetic big SAAS, little SAAS, PEN,
little LEN, and big LEN were determined by absorbance in a separate run.
Mass Spectrometry--
Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) spectra were obtained in positive
linear mode on a Perseptive Biosystems Voyager-DE STR mass spectrometer
(Framingham, MA). Approximately 100 laser shots were summed per
spectrum. Characterization of the Antisera against ProSAAS-derived
Peptides--
The antisera used in this study were raised against
three peptides derived from different regions of proSAAS. Little SAAS (proSAAS-(42-59)) represents an 18-residue peptide that is derived from the N-terminal region; PEN (proSAAS-(221-242)) is a 22-residue peptide from the C-terminal region; and big LEN (proSAAS-(245-260)) is
a 16-residue peptide and includes the C terminus of proSAAS (Fig.
1). All of the antisera raised in rabbit
exhibit reasonably high sensitivity toward the immunogenic peptide
(Table I). To characterize the
specificity of these antisera, we used a number of synthetic peptides
and glutathione S-transferase fusion proteins that contained
various regions of proSAAS (Table I). The anti-SAAS antibody recognizes
proSAAS-(34-59) (designated "big SAAS") and little SAAS but does
not recognize proSAAS-(34-40) (designated "KEP"). This antiserum
also recognizes the full-length proSAAS, albeit at somewhat reduced
levels (~30%), suggesting that the anti-SAAS antibody is directed to
the middle portion of little SAAS and that it is able to recognize N-
and C-terminally extended peptides with fairly high efficiency. The
anti-PEN antibody is also able to recognize C-terminally extended
peptides such as proSAAS-(221-260) ("big PEN-LEN") or
proSAAS-(221-254) ("little PEN-LEN"). In contrast, the anti-LEN
antibody recognizes only big LEN but not the C-terminally truncated
peptide, proSAAS-(245-254) ("little LEN") (Table I). These results
suggest that the epitope for this antiserum includes the C-terminal 6 residues of proSAAS. This is confirmed by the inability of anti-LEN
antibody to recognize little PEN-LEN, although it is able to recognize
big PEN-LEN (Table I). Taken together, these results indicate that the
three antisera recognize different domains of proSAAS and that they are
able to recognize the processed peptides as well as partially processed peptides and the unprocessed precursor.
The relative levels of proSAAS-derived peptides were measured in
various regions of mouse brain and pituitary. The highest levels of all
three peptides are found in the hypothalamus; the other brain regions
contain 3-30-fold lower levels (Table
II). The relative levels of these
peptides vary between brain regions. For example, the levels of
immunoreactive SAAS are comparable in pons/medulla and striatum;
however, the levels of immunoreactive PEN are substantially higher in
pons/medulla (Table II). Similarly, in AL and NIL of the pituitary, the
levels of immunoreactive PEN are substantially higher than the levels
of the other two peptides. In other areas, such as striatum and NIL,
the levels of immunoreactive LEN are substantially lower than the
levels of the other two peptides (Table II). These differences could be
due to the differential processing of the precursor in these tissues
leading to the generation of peptides that are recognized by the
antisera to different extents. Taken together, these results suggest
that proSAAS is differentially processed in various regions of the
brain and pituitary.
To examine the extent of processing of the precursor in the brain, the
extract from brains of Balb/c mice was subjected to gel filtration
chromatography followed by RIA with the antisera described above. We
find a broad peak of immunoreactive SAAS eluting between 3.5 and 1.9 kDa (Fig. 2). This could represent a
mixture of big SAAS, little SAAS, and/or partially processed
SAAS-containing higher molecular weight peptides. The immunoreactive
PEN also elutes as a broad peak between 3.2 and 1.9 kDa; this could
represent fully processed PEN and other PEN-containing peptides (such
as little PEN-LEN). In contrast, immunoreactive LEN elutes as a sharp peak at ~2 kDa (Fig. 2). Because the anti-LEN antibody does not recognize little LEN, it is likely that this immunoreactive peptide represents big LEN. Taken together, these results indicate that proSAAS
is processed to lower molecular weight peptides in mouse brain.
To further characterize these peptides, pools of fractions representing
peaks of immunoreactivity (fractions 24-27) were subjected to
reverse-phase HPLC under conditions that separate the fully processed
peptides from each other. The majority of immunoreactive SAAS elutes at
the position of little SAAS (Fig. 3).
Similar HPLC analysis of pools of fractions 22 and 23 also revealed
only little SAAS and no big SAAS (not shown), supporting the notion
that the major immunoreactive SAAS in mouse brain is little SAAS.
Immunoreactive PEN elutes as four peaks, one of which corresponds to
PEN (Fig. 3). The other three peaks could correspond to N- or
C-terminally extended PEN-containing peptides or to
post-translationally modified PEN. Because LEN immunoreactivity is not
detected in these fractions, it is unlikely that these peptides
represent big PEN-LEN, although little PEN-LEN (which is not detected
by the anti-LEN antiserum) may be present. Immunoreactive LEN elutes at
the position of big LEN (Fig. 3); little LEN could not be detected in
our system, since it does not contain the epitope recognized by the LEN
antiserum.
To confirm the identity of these peptides, fractions that contained the
peaks of immunoreactivity following HPLC were subjected to mass
spectrometry using both MALDI-TOF and ESI-TOF. The MALDI-TOF spectrum of HPLC fraction 37 shows a number of ions in the mass range 1335-4936 (Fig. 4). Because the
sample represents only gel filtration and reverse-phase HPLC, it is
expected to contain many peptides. A peak at 1755.98 is detected as a
moderately strong signal. This is precisely the calculated monoisotopic
mass of protonated big LEN. The ion is much weaker in fractions 36 and 38 and is completely absent from the other spectra analyzed. Similar MALDI-TOF analysis of HPLC fractions 30-58 did not reveal any close
similarities to the predicted masses of other proSAAS fragments, although some of these fractions contained such a large number of other
ions that a very strong signal for the proSAAS fragment would have been
required for detection. To reduce the problems of ion suppression that
occur when many ions are present, some of the fractions were analyzed
by ESI-TOF. HPLC fraction 43, which contains immunoreactive SAAS, was
found by ESI-TOF to contain a small but detectable peak at 907.09 (Fig.
4B). This peak is doubly protonated, based on analysis of
the enlarged spectra (Fig. 4B, inset) and
corresponds to an uncharged monoisotopic parent mass of 1812.18. This
mass is within 0.01% of the predicted mass of little SAAS (1812.00).
Taken together with the elution pattern of the immunoreactive peptides,
this is strong evidence that big LEN and little SAAS represent the
immunoreactive material detected in the HPLC fractions and that these
are the major fragments of these peptides in mouse brain.
Previous studies have found that the endoproteolytic processing of a
number of peptide hormones are substantially decreased in
Cpefat/Cpefat mice (12-15). To
examine if the processing of proSAAS is altered in these animals, brain
extracts from Cpefat/Cpefat and
wild-type littermates were subjected to gel filtration chromatography followed by RIA. In the
Cpefat/Cpefat mouse brain, there
is a reduction in the level of processed lower molecular weight
immunoreactive SAAS peptides and a small increase in the partially
processed higher molecular weight forms (Fig. 5). There is also a decrease in the
levels of immunoreactive PEN and a shift to a slightly higher molecular
weight form in the mice lacking CPE activity (Fig. 5). In contrast to
immunoreactive PEN and immunoreactive SAAS, there is an increase in the
level of immunoreactive LEN in these mice (Fig. 5). Because the
anti-LEN antiserum recognizes only big LEN and not little LEN, these
results suggest that the proteolytic cleavage that generates little LEN is not as efficient in
Cpefat/Cpefat mice compared with
control mice.
The processing profile of proSAAS was also examined in the AL and the
NIL of the pituitary. The gel filtration profile of immunoreactive SAAS
is altered in the NIL of
Cpefat/Cpefat mice, with a slight
accumulation of higher molecular weight SAAS-containing intermediate
forms (Fig. 6, top). In AL,
there is a small shift in the size of immunoreactive SAAS in the
Cpefat/Cpefat mice, possibly
reflecting the accumulation of Arg-extended peptide (Fig. 6,
top). There is also a small shift in the molecular weight of
immunoreactive PEN in the AL of
Cpefat/Cpefat but no substantial
change in the NIL (Fig. 6, middle). The level of immunoreactive LEN in
AL is comparable between
Cpefat/Cpefat and wild type mice,
whereas there is a substantial increase in the level of immunoreactive
LEN in NIL of Cpefat/Cpefat mice
as compared with wild type mice (Fig. 6, bottom). These results suggest a decrease in the processing of LEN in NIL of these
mice, leading to the accumulation of big LEN. Thus, it appears that in
addition to the defect in carboxypeptidase activity in the
Cpefat/Cpefat mice, these mice
also have a defect in endoproteolytic activity that leads to small
changes in the processing of proSAAS by endopeptidases.
ProSAAS is a recently characterized protein with some
characteristics in common with the granins (16). Although there is no
amino acid sequence homology between granins, except for between chromogranins A and B, the granins are considered a family because they
are all broadly expressed neuroendocrine proteins that are secreted via
the regulated pathway. In addition, the granins contain a relatively
high abundance of acidic residues, and they also contain multiple pairs
of basic amino acids. However, most of the granins are not processed
extensively at these pairs of basic residues, raising doubts as to
whether these proteins are primarily precursors of neuroendocrine
peptides. Although proSAAS generally fits these loose criteria of
granins, our finding that proSAAS is extensively processed in brain and
pituitary argues against this protein being a chromogranin A- or B-like
granin and instead suggests that this protein functions as a
neuroendocrine peptide precursor.
Peptides derived from proSAAS were previously identified in
Cpefat/Cpefat mice using a novel
approach to isolate incorrectly processed peptides (16). However, the
techniques previously used could not determine whether these processed
peptides represented major or minor forms of proSAAS. The gel
filtration and RIA methods used in the present study are quantitative
and complement the previous analyses. A concern with RIAs is that the
antisera may not be specific for the peptide or protein; if so, the
detected signal may be due to some other peptide or protein. In the
present study, this is unlikely for several reasons. First, our RIA
data show high levels of SAAS, PEN, and LEN in the hypothalamus,
consistent with the distribution of proSAAS mRNA as determined by
Northern blot analysis and by in situ hybridization (16).
The generally similar pattern of expression of all three peptides
further validates the RIA. Second, the use of gel filtration and
reverse-phase HPLC to show coelution of the immunoreactive SAAS and
immunoreactive LEN with little SAAS and big LEN, respectively, is
further evidence that these peptides are responsible for the RIA
signal. Finally, the finding that peptides with the mass of little SAAS
and big LEN are present in the appropriate HPLC fractions is strong
confirmation that these peptides are produced in wild-type mouse brain.
The reduced level in brain and NIL of immunoreactive LEN (representing
big LEN) in wild type as compared with
Cpefat/Cpefat mice suggests a
role for PC1 or PC2 in the generation of little LEN; both of these
enzymes are thought to be less active in the Cpefat/Cpefat mice compared with
wild type mice (23). Because the difference in immunoreactive LEN
levels between the Cpefat/Cpefat
and control animals is much greater in the NIL than in the AL, it is
likely that PC2 is primarily involved in processing big LEN into little
LEN. PC2 is abundant in the intermediate pituitary and is thought to
process ProSAAS has been shown to be a potent inhibitor of PC1 (16). Recent
studies by two independent groups have narrowed down the PC1-inhibitory
region to a short region of proSAAS near the junction of PEN and LEN
(17, 18). While the fully processed form of PEN is not inhibitory (17),
the Lys-Arg-extended PEN peptide inhibits PC1 activity (18). In
Cpefat/Cpefat mice, the lack of
CPE activity results in the accumulation of PEN-Lys-Arg (16); this
peptide may affect the autocatalytic maturation of PC1. Consistent with
this possibility, the maturation and activity of PC1 are decreased in
Cpefat/Cpefat (23).
In previous studies using mass spectrometry and immunoprecipitation to
examine the processing of proSAAS in AtT-20 cells overexpressing this
protein, several products were detected including big SAAS, little
SAAS, PEN, big LEN, little PEN/LEN, and big PEN/LEN. In the present
study, wild type mice brains were found to have substantial amounts of
little SAAS, PEN, and big LEN, but not big SAAS, big PEN/LEN, or larger
forms of these peptides. Thus, while PEN/LEN and larger forms of this
peptide are inhibitory to PC1, the finding that the major forms in wild
type mouse brain and pituitary are the smaller noninhibitory forms
suggests that PC1 is not tonically inhibited by the proSAAS peptides.
Furthermore, the extensive processing of proSAAS implies that this
protein is a neuropeptide precursor. While any peptide produced in
neural tissue could be considered a "neuropeptide," this term is
usually reserved for peptides that function in signaling between cells.
The criteria for establishing a peptide as a signaling molecule include
demonstrating that it is produced in neuroendocrine cells, that it is
secreted upon stimulation, and that it binds to a cellular receptor,
causing changes in second messengers. The present results are strong
evidence that little SAAS and big LEN are produced in brain and
pituitary, and thus are candidate "neuropeptides." Previous studies
have shown that in AtT-20 cells, proSAAS is routed into the regulated secretory pathway (16), and recent studies have shown that the immunoreactive SAAS and immunoreactive LEN are secreted upon
stimulation.2 However, it is
essential that a receptor for the peptides be found before they can be
considered signaling molecules. Now, with the evidence presented here
that little SAAS and big LEN are major products of proSAAS in brain, it
is possible to begin searching for specific receptors for these peptides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amidating monooxygenase (4).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C until use.
20 °C. Prior to RIAs and/or
gel exclusion chromatography, samples from both extractions were
resuspended in sodium phosphate buffer, pH 7.5, containing 0.2% Triton
X-100 and pooled.
-Cyano-4-hydroxycinnamic acid saturated in 30%
acetonitrile and 0.1% trifluoroacetic acid in water was used as
a matrix. External calibration was performed with
des-Arg1-bradykinin ([M + H]+ = 904.4681) and
neurotensin ([M + H]+ = 1672.9170). Electrospray
time-of-flight (ESI-TOF) spectra were recorded on a Perseptive
Biosystems Mariner mass spectrometer (Framingham, MA). Samples were
introduced directly into the Mariner by infusion. System tuning and
mass calibration were performed with angiotensin I ([M + H]+ = 1296.6853, [M + 2H]2+ = 648.8466, and
[M + 3H]3+ = 432.9003).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 1.
Schematic representation of proSAAS and known
sites of endoproteolytic cleavage. The proSAAS-derived peptides
and their relative positions within the precursor are shown in
boxes below. Basic processing sites are
indicated: arginine (R), lysine (K), and signal
peptide (SP). Fusion constructs consisting of glutathione
S-transferase and C-terminal extensions of the indicated
portions of rat proSAAS are shown as lines.
Characterization of antisera against SAAS, PEN, and LEN peptides
ProSAAS-derived peptides in mouse brain regions and pituitary
View larger version (19K):
[in a new window]
Fig. 2.
Fractionation of mouse brain extracts by gel
filtration chromatography and analysis of immunoreactive SAAS,
immunoreactive PEN, and immunoreactive LEN. Extracts from three
mouse brains were pooled, and a portion of the material was subjected
to gel filtration chromatography on Superdex Peptide 10/30 column as
described. The fractions were analyzed for immunoreactive SAAS,
immunoreactive PEN, and immunoreactive LEN as described under
"Materials and Methods." Molecular mass calibration
standards are as follows: cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; -endorphin, 3.5 kDa;
-MSH, 1.7 kDa; Dyn A-8, 1.0 kDa.
ir-peptide, immunoreactive peptide.
View larger version (22K):
[in a new window]
Fig. 3.
HPLC analysis of the peak of immunoreactive
proSAAS-derived peptides following gel filtration analysis.
Top, schematic representation of proSAAS precursor showing
possible single and paired basic cleavage sites as well as products of
post-translational processing. R, arginine; K,
lysine, L.SAAS, little SAAS; B.SAAS, big SAAS;
L.LEN, little LEN; B.LEN, big LEN.
Bottom, fractions representing the peak of immunoreactive
SAAS, immunoreactive PEN, and immunoreactive LEN were pooled, and a
portion of the material was subjected to HPLC on a C18
column. A linear gradient of acetonitrile from 0 to 75% was used; on
this gradient, the synthetic peptides were found to be well separated.
The fractions were analyzed for immunoreactive SAAS (top),
immunoreactive PEN (middle), and immunoreactive LEN
(bottom) as described. The lines at the
top denote the elution positions of synthetic peptides
(B, big form; L, little form).
ir-peptide, immunoreactive peptide.
View larger version (32K):
[in a new window]
Fig. 4.
A, MALDI-TOF mass spectrum of
reverse-phase HPLC fraction 37. The peak marked by an
asterisk represents positively charged big LEN. The expected
and the observed monoisotopic [MH]+ mass are both
1755.98. B, ESI-TOF mass spectrum of reverse-phase HPLC
fraction 43. The peak marked by an asterisk
represents doubly protonated little SAAS. The inset shows
the enlarged spectrum of the peak with a monoisotopic mass/charge of
907.09 and a charge state of 2+ (uncharged parent mass = 1812.18). The expected monoisotopic mass of little SAAS is 1812.00 (m/z = 907.00 for the 2+
ion).
View larger version (18K):
[in a new window]
Fig. 5.
Fractionation of wild type (solid
line) and
Cpefat/Cpefat
(dashed line) mouse brain extracts by gel
filtration chromatography and analysis of immunoreactive SAAS,
immunoreactive PEN, and immunoreactive LEN. Extracts
from three mouse brains were pooled, and a portion of the material was
subjected to gel filtration chromatography on Superdex Peptide 10/30
column as described. The fractions were analyzed for immunoreactive
SAAS (top), immunoreactive PEN (middle), and
immunoreactive LEN (bottom) as described under "Materials
and Methods." Molecular mass calibration standards are as follows:
cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; -endorphin, 3.5 kDa;
-MSH, 1.7 kDa; Dyn A-8, 1.0 kDa. ir-peptide,
immunoreactive peptide.
View larger version (25K):
[in a new window]
Fig. 6.
Fractionation of peptides from the anterior
(left) and neurointermediate (right)
lobes of the pituitary from wild type mice (solid
line) and
Cpefat/Cpefat mice
(dashed line) by gel filtration
chromatography on a Superdex Peptide 10/30 column. Top,
schematic representation of proSAAS precursor showing possible single
and paired basic cleavage sites as well as products of
post-translational processing. R, arginine; K,
lysine, L.SAAS, little SAAS; B.SAAS, big SAAS;
L.LEN, little LEN; B.LEN, big LEN.
Bottom, the fractions were analyzed for immunoreactive SAAS
(top), immunoreactive PEN (middle), and
immunoreactive LEN (bottom) as described under "Materials
and Methods." Molecular mass calibration standards are as follows:
cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; -endorphin, 3.5 kDa;
-MSH, 1.7 kDa; Dyn A-8, 1.0 kDa. ir-peptide,
immunoreactive peptide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lipotropin to
-endorphin and to cleave adrenocorticotropic hormone into the
-melanocyte-stimulating hormone
precursor (24, 25). Recent studies in transgenic animals lacking PC2
have shown an involvement of this enzyme in the generation of small
bioactive peptide hormones and neuropeptides (26-29). Thus, a role for
PC2 in processing big LEN into little LEN would be consistent with this
enzyme's actions on other peptides.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. James Douglass for providing antisera and some of the peptides and Haiteng Deng for assistance with the Mariner mass spectrometry, which was performed in the Laboratory of Macromolecular Analysis of the Albert Einstein College of Medicine.
![]() |
FOOTNOTES |
---|
* This work is supported in part by National Institutes of Health Grants R01-NS26880 and K02-DA00458 (to L. A. D.), K01-DA00342 (to Y. B.), and R01-DA04494 and K02-DA00194 (to L. D. F.).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: Dept. of Pharmacology, New York University Medical Center, MSB 411, 550 First Ave., New York, NY 10016. Tel.: 212-263-7119; Fax: 212-263-7133; E-mail: Lakshmi.Devi@med.nyu.edu.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M009067200
2 N. Mzhavia and L. A. Devi, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PC, prohormone
convertase;
CPE, carboxypeptidase E;
RIA, radioimmunoassay;
AL, anterior lobe;
NIL, neurointermediate lobe;
HPLC, high performance
liquid chromatography;
ESI-TOF, electrospray time-of-flight;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass
spectrometry;
ACTH, adrenocorticotropic hormone;
-MSH,
-melanocyte-stimulating hormone.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Docherty, K., and Steiner, D. F. (1982) Annu. Rev. Physiol. 44, 625-638[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Zhou, A.,
Webb, G.,
Zhu, X.,
and Steiner, D. F.
(1999)
J. Biol. Chem.
274,
20745-20748 |
3. | Fricker, L. D. (1988) Annu. Rev. Physiol. 50, 309-321[CrossRef][Medline] [Order article via Infotrieve] |
4. | Mains, R. E., Dickerson, I. M., May, V., Stoffers, D. A., Perkins, S. N., Ouafik, L., Husten, E. J., and Eipper, B. A. (1990) Front. Neuroendocrinol. 11, 52-89 |
5. | Seidah, N. G., and Chretien, M. (1997) Curr. Opin. Biotechnol. 8, 602-607[CrossRef][Medline] [Order article via Infotrieve] |
6. | Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. P., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 297-5301 |
7. |
Steiner, D. F.,
Smeekens, S. P.,
Ohagi, S.,
and Chan, S. J.
(1992)
J. Biol. Chem.
267,
23435-23438 |
8. |
Bergeron, F.,
Leduc, R.,
and Day, R.
(2000)
J. Mol. Endocrinol.
24,
1-22 |
9. | Seidah, N. G., Day, R., Marcinkiewicz, M., Benjannet, S., and Chretien, M. (1991) Enzyme 45, 271-284[Medline] [Order article via Infotrieve] |
10. |
Seidah, N. G.,
Day, R.,
Marcinkiewicz, M.,
and Chretien, M.
(1998)
Ann. N. Y. Acad. Sci.
839,
9-24 |
11. | Schafer, M. K.-H., Day, R., Cullinan, W. E., Chretien, M., Seidah, N. G., and Watson, S. J. (1993) J. Neurosci. 13, 1258-1279[Abstract] |
12. | Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142[Medline] [Order article via Infotrieve] |
13. |
Fricker, L. D.,
Berman, Y. L.,
Leiter, E. H.,
and Devi, L. A.
(1996)
J. Biol. Chem.
271,
30619-30624 |
14. | Fricker, L. D., and Leiter, E. H. (1999) Trends Biochem. Sci. 24, 390-393[CrossRef][Medline] [Order article via Infotrieve] |
15. | Rovere, C., Viale, A., Nahon, J-L., and Kitabgi, P. (1996) Endocrinology 137, 2954-2958[Abstract] |
16. |
Fricker, L. D.,
McKinzie, A. A.,
Sun, J.,
Curran, E.,
Qian, Y.,
Yan, L.,
Patterson, S. D.,
Courchesne, P. L.,
Richards, B.,
Levin, N.,
Mzhavia, N.,
Devi, L. A.,
and Douglass, J.
(2000)
J. Neurosci.
20,
639-648 |
17. |
Qian, Y.,
Devi, L. A.,
Mzhavia, N.,
Munzer, S.,
Seidah, N. G.,
and Fricker, L. D.
(2000)
J. Biol. Chem.
275,
23596-23601 |
18. | Cameron, A., Fortenberry, Y., and Lindberg, I. (2000) FEBS Lett. 473, 135-138[CrossRef][Medline] [Order article via Infotrieve] |
19. | Muller, L., and Lindberg, I. (1999) Prog. Nucleic Acids Res. Mol. Biol. 63, 69-108[Medline] [Order article via Infotrieve] |
20. | Natori, S., and Huttner, W. B. (2000) Biochimie (Paris) 76, 277-282 |
21. | Li, J-Y., Leitner, B., Lovisetti-Scamihorn, P., Winkler, H., and Dahlstrom, A. (1999) Eur. J. Neurosci. 11, 528-544[CrossRef][Medline] [Order article via Infotrieve] |
22. | Glowinski, J., and Iversen, L. L. (1966) J. Neurochem. 13, 655-669[Medline] [Order article via Infotrieve] |
23. |
Berman, Y.,
Mzhavia, N.,
Polonskaia, A.,
and Devi, L. A.
(2001)
J. Biol. Chem.
276,
1466-1473 |
24. | Benjannet, S., Rondeau, N., Day, R., Chretien, M., and Seidah, N. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3564-3568[Abstract] |
25. | Day, R., Schafer, M. K., Watson, S. J., Chretien, M., and Seidah, N. G. (1992) Mol. Endocrinol. 6, 485-497[Abstract] |
26. |
Furuta, M.,
Carroll, R.,
Martin, S.,
Swift, H. H.,
Ravazzola, M.,
Orci, L.,
and Steiner, D. F.
(1998)
J. Biol. Chem.
273,
3431-3437 |
27. |
Day, R.,
Lazure, C.,
Basak, A.,
Boudreault, A.,
Limperist, P.,
Dong, W.,
and Lindberg, I.
(1998)
J. Biol. Chem.
273,
829-836 |
28. |
Johanning, K.,
Juliano, M. A.,
Juliano, L.,
Lazure, C.,
Lamango, N. S.,
Steiner, D. F.,
and Lindberg, I.
(1998)
J. Biol. Chem.
273,
22672-22680 |
29. | Berman, Y., Mzhavia, N., Polonskaia, A., Furuta, M., Steiner, D. F., Pintar, J. E., and Devi, L. A. (2000) J. Neurochem. 75, 1763-1770[CrossRef][Medline] [Order article via Infotrieve] |