ProSAAS Processing in Mouse Brain and Pituitary*

Nino Mzhavia, Yemiliya Berman, Fa-Yun CheDagger , Lloyd D. FrickerDagger , and Lakshmi A. Devi§

From the Department of Pharmacology, New York University School of Medicine, New York, New York 10016 and the Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -amidating monooxygenase (4).

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -80 °C until use.

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 -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.

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. alpha -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

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.



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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.


                              
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Table I
Characterization of antisera against SAAS, PEN, and LEN peptides
The data represent mean of triplicate determinations from two independent studies.

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.


                              
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Table II
ProSAAS-derived peptides in mouse brain regions and pituitary
The data represent mean ± S.E. from triplicate determinations with three animals; ND, not done; Ir, immunoreactive.

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.



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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; beta -endorphin, 3.5 kDa; alpha -MSH, 1.7 kDa; Dyn A-8, 1.0 kDa. ir-peptide, immunoreactive peptide.

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.



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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.

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.



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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).

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.



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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; beta -endorphin, 3.5 kDa; alpha -MSH, 1.7 kDa; Dyn A-8, 1.0 kDa. ir-peptide, immunoreactive peptide.

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.



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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; beta -endorphin, 3.5 kDa; alpha -MSH, 1.7 kDa; Dyn A-8, 1.0 kDa. ir-peptide, immunoreactive peptide.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -lipotropin to beta -endorphin and to cleave adrenocorticotropic hormone into the alpha -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.

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.


    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; alpha -MSH, alpha -melanocyte-stimulating hormone.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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