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• W1995934769 abstract "ProSAAS is a recently discovered 26-kDa neuroendocrine protein that was previously found to inhibit prohormone convertase (PC) 1 and not PC2. In the present study, the specificity of proSAAS toward other members of the prohormone convertase family was determined. Two μm proSAAS selectively inhibits PC1 but not furin, PACE4, PC5A, or PC7. The PC1 inhibitory region of proSAAS was mapped to an 8–12-residue region near the C terminus that includes a critical Lys-Arg sequence. Synthetic peptides corresponding to this region are competitive inhibitors of PC1 with apparentKi values of 14–40 nm. The inhibition becomes more effective with incubation time, indicating that the inhibitor is slow binding. A fusion protein containing the inhibitory region of proSAAS linked to the C terminus of glutathioneS-transferase binds the 71-kDa form but not the 85-kDa form of PC1. This binding, which occurs at pH 5.5 and not at pH 7.4, is stable to incubation at room temperature for 1 h in the presence or absence of 0.5% Triton X-100 and/or 0.5 mNaCl. The removal of Ca2+ with chelating agents partially releases the bound PC1. High concentrations of the inhibitory peptide quantitatively release the bound PC1. Taken together, these data support the proposal that proSAAS functions as an endogenous inhibitor of PC1. ProSAAS is a recently discovered 26-kDa neuroendocrine protein that was previously found to inhibit prohormone convertase (PC) 1 and not PC2. In the present study, the specificity of proSAAS toward other members of the prohormone convertase family was determined. Two μm proSAAS selectively inhibits PC1 but not furin, PACE4, PC5A, or PC7. The PC1 inhibitory region of proSAAS was mapped to an 8–12-residue region near the C terminus that includes a critical Lys-Arg sequence. Synthetic peptides corresponding to this region are competitive inhibitors of PC1 with apparentKi values of 14–40 nm. The inhibition becomes more effective with incubation time, indicating that the inhibitor is slow binding. A fusion protein containing the inhibitory region of proSAAS linked to the C terminus of glutathioneS-transferase binds the 71-kDa form but not the 85-kDa form of PC1. This binding, which occurs at pH 5.5 and not at pH 7.4, is stable to incubation at room temperature for 1 h in the presence or absence of 0.5% Triton X-100 and/or 0.5 mNaCl. The removal of Ca2+ with chelating agents partially releases the bound PC1. High concentrations of the inhibitory peptide quantitatively release the bound PC1. Taken together, these data support the proposal that proSAAS functions as an endogenous inhibitor of PC1. prohormone convertase glutathione S-transferase 7-amino-4-methylcoumarin Most bioactive peptides are produced from larger precursors by selective cleavage at specific sites (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Typically, these sites contain basic amino acids with the consensus (K/R)Xn R where n = 0, 2, 4, or 6 and X indicates any amino acid(s) except cysteine (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar). Processing at these sites is accomplished by an endopeptidase that cleaves to the C-terminal side of the consensus site and then a carboxypeptidase to remove the C-terminal basic residue(s). The prohormone/proprotein convertases (PCs)1 are a family of mammalian endopeptidases that are related to bacterial subtilisin and to the yeast KEX2 endopeptidase (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar). The PCs can be divided into two subgroups based on their distribution within cells. One group, which includes furin, PACE4, PC5B (also known as PC6B), and PC7 (also known as PC8 and LPC), is primarily localized to the trans-Golgi network in a variety of cell types (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar). The other group, which includes PC1 (also known as PC3), PC2, and PC5A (also known as PC6A), is enriched in secretory vesicles in neuroendocrine cell types (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar). In contrast to the multitude of PCs, only two carboxypeptidases are thought to be involved in processing proteins in the secretory pathway. Carboxypeptidase D is primarily localized to the trans-Golgi network, and carboxypeptidase E is enriched in secretory vesicles (3Varlamov O. Fricker L.D. J. Cell Sci. 1998; 111: 877-885Crossref PubMed Google Scholar,4Varlamov O. Eng F.J. Novikova E.G. Fricker L.D. J. Biol. Chem. 1999; 274: 14759-14767Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The enzymes localized to the trans-Golgi network are thought to process proteins that transit the constitutive secretory pathway, such as the insulin receptor and some growth factors (5Bresnahan P.A. Leduc R. Thomas L. Thorner J. Gibson H.L. Brake A.J. Barr P.J. Thomas G. J. Cell Biol. 1990; 111: 2851-2859Crossref PubMed Scopus (288) Google Scholar, 6Bravo D.A. Gleason J.B. Sanchez R.I. Roth R.A. Fuller R.S. J. Biol. Chem. 1994; 269: 25830-25837Abstract Full Text PDF PubMed Google Scholar, 7Komada M. Hatsuzawa K. Shibamoto S. Ito F. Nakayama K. Kitamura N. FEBS Lett. 1993; 328: 25-29Crossref PubMed Scopus (108) Google Scholar). The secretory vesicle enzymes are primarily involved in the processing of neuroendocrine peptides (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar).A large number of enzyme systems have endogenous inhibitors that serve to regulate the enzyme activity. All of the PCs are initially produced as zymogens that must be proteolytically activated (1Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (240) Google Scholar). In some cases, the pro-segment is still an effective inhibitor even after initial proteolysis and additional cleavages are required to fully activate the enzyme (8Boudreault A. Gauthier D. Lazure C. J. Biol. Chem. 1998; 273: 31574-31580Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 9Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chretien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In addition to being produced as a pro-form, PC2 is also co-expressed with an endogenous chaperone/inhibitor, 7B2 (10Braks J.A.M. Martens G.J.M. Cell. 1994; 78: 263-273Abstract Full Text PDF PubMed Scopus (195) Google Scholar). 7B2 was initially discovered as a granin-like protein present in the secretory pathway of neuroendocrine cells (11Hsi K.L. Seidah N.G. DeSerres G. Chretien M. FEBS Lett. 1982; 147: 261-266Crossref PubMed Scopus (117) Google Scholar). This protein is initially produced as a 27–30-kDa protein that is proteolytically processed by furin (12Paquet L. Bergeron F. Boudreault A. Seidah N.G. Chretien M. Mbikay M. Lazure C. J. Biol. Chem. 1994; 269: 19279-19285Abstract Full Text PDF PubMed Google Scholar) into a 31-residue C-terminal peptide (13Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Crossref PubMed Scopus (76) Google Scholar). Although co-expression of 7B2 is necessary for the production of active PC2 (14Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar), the C-terminal peptide of 7B2 is also a potent inhibitor of PC2 (13Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Crossref PubMed Scopus (76) Google Scholar, 15van Horssen A.M. van den Hurk W.H. Bailyes E.M. Hutton J.C. Martens G.J. Lindberg I. J. Biol. Chem. 1995; 270: 14292-14296Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PC2 slowly cleaves at a Lys-Lys site within this C-terminal peptide, and then, following the action of a carboxypeptidase, the cleaved peptide is no longer inhibitory toward PC2 (13Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Crossref PubMed Scopus (76) Google Scholar).Recently, a novel secretory pathway protein was detected while analyzing peptides that were not properly processed in mice lacking carboxypeptidase E activity (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). The Cpe fat /Cpe fat mice contain a point mutation in the carboxypeptidase E gene that renders the enzyme inactive (17Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Crossref PubMed Scopus (607) Google Scholar). Without active carboxypeptidase E, these mice accumulate peptides with C-terminal Lys and/or Arg extensions (17Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Crossref PubMed Scopus (607) Google Scholar, 18Fricker L.D. Berman Y.L. Leiter E.H. Devi L.A. J. Biol. Chem. 1996; 271: 30619-30624Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Rovere C. Viale A. Nahon J. Kitabgi P. Endocrinology. 1996; 137: 2954-2958Crossref PubMed Scopus (124) Google Scholar). A large number of improperly processed peptides were detected using an affinity column to isolate peptides with C-terminal basic residues fromCpe fat /Cpe fat tissues (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). Mass spectrometry-based sequence analysis revealed that five of these peptides were encoded by a novel protein, designated proSAAS (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). Although there is no amino acid sequence homology between proSAAS and 7B2, they are both similar sizes, they both contain a relatively high percentage of proline as well as several pairs of basic amino acids, and they both have a broad neuroendocrine tissue distribution (10Braks J.A.M. Martens G.J.M. Cell. 1994; 78: 263-273Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 11Hsi K.L. Seidah N.G. DeSerres G. Chretien M. FEBS Lett. 1982; 147: 261-266Crossref PubMed Scopus (117) Google Scholar, 16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar, 20Marcinkiewicz M. Benjannet S. Seidah N.G. Cantin M. Chretien M. Cell Tissue Res. 1987; 250: 205-214Crossref PubMed Scopus (51) Google Scholar). Based on this similarity and on the finding that overexpression of proSAAS in AtT-20 cells inhibited the processing of the endogenous prohormone, proopiomelanocortin, proSAAS was directly tested with PC1 and PC2 (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). This analysis revealed that proSAAS inhibited PC1 but not PC2. The specificity of proSAAS for other members of the PC family was not previously investigated. In this study, we report that proSAAS is highly specific for PC1 and does not inhibit other members of this gene family. In addition, we map the inhibitory region to a short 8–12-amino acid segment near the C terminus of proSAAS. Peptides corresponding to this region inhibit PC1 with a slow onset of action that is partially competitive with substrate. Finally, this region of proSAAS is able to bind PC1 tightly with a slow dissociation rate. Taken together, these data support the proposal that one of the physiological functions of proSAAS is to inhibit PC1.RESULTSPreviously, rat proSAAS produced as a GST fusion protein in bacteria was found to inhibit PC1 (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). To assess the specificity of proSAAS for PC1, the GST-proSAAS fusion protein was tested with other members of the PC family. At 2 μm concentration the GST-proSAAS does not significantly inhibit PC2, furin, PACE4, PC5, or PC7 (Table I). Although a small decrease in PC2 activity was observed, this decrease was not significantly different from the activity in the presence of GST alone (Table I). In addition, kexin activity was not significantly affected by this concentration of GST-proSAAS (not shown).Table IEffect of 2 μm GST-proSAAS or GST on the activity of various members of the prohormone convertase familyEnzymeActivity1-aValues are the averages of three separate experiments, each performed in duplicate using 100 μm pyroGlu-Arg-Thr-Lys-Arg-AMC substrate.GST aloneGST-proSAAS% control ± S.D.PC176 ± 420 ± 1PC283 ± 1070 ± 8Furin103 ± 2101 ± 3PACE4101 ± 2105 ± 4PC5A103 ± 3102 ± 4PC799 ± 397 ± 41-a Values are the averages of three separate experiments, each performed in duplicate using 100 μm pyroGlu-Arg-Thr-Lys-Arg-AMC substrate. Open table in a new tab To map the inhibitory region of rat proSAAS, a series of deletion mutants was expressed as GST fusion proteins and tested with baculovirus-expressed PC1. Deletion of the C-terminal 28 residues abolished the effectiveness of proSAAS as a PC1 inhibitor (Fig.1, 34–232). Conversely, constructs that contained the C-terminal 30 residues (Fig. 1,231–260 and others) were inhibitory. Further deletion analysis indicated that the 16-residue region from residues 231–246 of rat proSAAS were inhibitory (Fig. 1); this region contains an internal Lys-Arg sequence that was previously found to be cleaved in AtT-20 cells and in mouse brain (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). Synthetic peptides corresponding to the cleavage products were ineffective as PC1 inhibitors (Fig. 1). The inhibitory activity of the 16-residue proSAAS C-terminal peptide is not dependent on attachment to GST; cleavage with thrombin did not alter the inhibitory potency of the construct. Mutation of both the Lys243 and Arg244 to Ala within the construct containing residues 231–246 of proSAAS destroyed the ability of this fragment to inhibit PC1 activity (Fig. 1).To further map the inhibitory region, a series of peptides was synthesized. A 17-residue peptide corresponding to residues 231–246 of proSAAS with an additional Tyr on the N terminus inhibited PC1 with an IC50 of approximately 35 nm (Fig.2, YE17). The 14- and 12-residue peptides showed comparable potency toward PC1, whereas the 10- and 8-residue peptides had IC50 values around 70 nm (Fig. 2). To test whether this inhibition is due to a nonpeptide contaminant present in the peptide, the peptides were treated with trypsin, the trypsin was then heat-inactivated, and the peptides were tested with PC1. This trypsin treatment eliminated the inhibitory activity of the peptides toward PC1, indicating that the inhibition was due to a peptide component (and also supporting the data shown in Fig. 1 illustrating the importance of the intact Lys-Arg sequence for PC1 inhibition). The specificity of YE-17 for various members of the PC family was evaluated. As found with full-length proSAAS, low concentrations (0.25–2 μm) of YE-17 selectively inhibit PC1. However, at 25 μm YE-17 some inhibition of PC7 and furin were detected and to a much lesser extent PC5 and PACE4 (not shown). It is possible that this inhibition observed with 25 μm YE-17 is due to competition with the 100 μm substrate.Figure 2Effect of synthetic proSAAS C-terminal peptides on the activity of purified PC1. The peptides were tested with purified PC1 (63 ng) and 100 μm substrate, as described under “Materials and Methods.” The data shown were obtained after 90 min of incubation at 25 °C. The experiment was performed three times with less than 10% variation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kinetic analysis of PC1 in the presence of various concentrations of YE-17 showed a mixture of noncompetitive and competitive-type inhibition with a Ki of 14 nm (Fig.3). Similar analysis of LE-8 showed pure competitive inhibition kinetics (not shown). The portion of inhibition that appears to be noncompetitive may be due to slow dissociation of YE-17 from the active site rather than true noncompetitive inhibition. Analysis of the time course of PC1 activity in the presence of various amounts of YE-17 showed that relatively high concentrations of peptide did not immediately inhibit PC1 (Fig. 4). Instead, the progress curves for the reaction followed typical slow binding kinetics, with greater inhibition observed at later reaction times (Fig. 4).Figure 3Double reciprocal plots of purified PC1 activity with two concentrations of YE-17. Purified PC1 (63 ng) was added to a mixture of the indicated concentrations of YE-17 and substrate, as described under “Materials and Methods.” The data were obtained after 55–90 min incubation at 25 °C. The experiment was performed three times with comparable results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Progress curves of PC1 activity in the presence of various concentrations of YE-17. Purified PC1 (63 ng) was added to 100 μm substrate and the indicated concentration of YE-17, and the reaction was followed for 50 min at room temperature.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test whether PC1 is able to bind tightly to the proSAAS C-terminal peptides, several GST-proSAAS constructs were incubated with media from Sf9 cells infected with PC1-expressing baculovirus and then the GST purified on glutathione-agarose. The resin was washed briefly with binding buffer, and the bound material was eluted by boiling in 1% SDS. When the binding was performed at pH 5.5, the 71-kDa form of PC1 in the baculovirus media bound to the full-length proSAAS construct (Fig. 5,lanes 2) and the construct containing proSAAS 221–260 (Fig. 5, lanes 3). No binding was detected to GST alone (Fig. 5, lanes 1) or to the construct containing proSAAS 34–174 (Fig. 5, lanes 4), which lacks the C-terminal PC1 inhibitory region. When binding was performed at pH 7.4, no binding was detected for any of the constructs or forms of PC1 (Fig. 5, bottom panel). Longer exposures of the Western blot revealed that in addition to the 71-kDa form, the less abundant 75-kDa form of PC1 in the media (but not the 85-kDa form) showed some binding to the full-length proSAAS and to proSAAS 221–260 at pH 5.5 but not to the other inactive constructs (not shown).Figure 5Western blot analysis of PC1 binding to GST-proSAAS constructs. Medium from Sf9 cells infected with PC1-expressing baculovirus was combined with either GST or a GST-proSAAS fusion protein at pH 5.5 (top panel) or pH 7.4 (bottom panel). Then glutathione-agarose was added, the tubes were mixed for 1 h and centrifuged, and the supernatant was removed for Western blot analysis (Unbound). The pellet was washed twice with 1.5 ml of binding buffer for 3 min each (Wash 1 and Wash 2). Then the pellet was heated at 95 °C in 1% SDS to extract the PC1 bound to the resin (Bound). The same relative amount of each fraction was analyzed on a Western blot that was probed with 1:1000 dilution of an antiserum directed against PC1. The positions of prestained molecular weight markers (Life Technologies, Inc.) are indicated. Lane S, stock of PC1-expressing baculovirus medium; lane 1, GST alone;lane 2, GST-proSAAS 34–260; lane 3, GST-proSAAS 221–260; lane 4, GST-proSAAS 34–174.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate whether the binding is competitive with peptide YE-17, PC1 was incubated with GST-proSAAS construct 221–246, and the material was isolated on glutathione-agarose and then incubated for 1 h at room temperature under various conditions. In the presence of buffer alone, all of the PC1 remained bound to the GST-proSAAS construct (Fig.6). The addition of 0.5 mNaCl and 0.5% Triton X-100 did not release the bound PC1 (Fig. 6,lanes Tx). The calcium chelator EDTA was partially effective at releasing the bound PC1 (Fig. 6). The peptide YE-17 quantitatively released the bound PC1 when tested at either 100 or 300 μm (Fig. 6), but 1 μm peptide was ineffective (not shown). Approximately 50% of the bound PC1 was released by the peptide within 10 min of incubation (Fig.6 C). However, incubation for 70 min with 100 μm of the PC1 substrate pGlu-Arg-Thr-Lys-Arg-MCA was not effective in releasing bound PC1 (Fig. 6 C).Figure 6Western blot analysis of release of proSAAS-bound PC1. A and B, medium from PC1-expressing baculovirus was combined with GST-proSAAS 221–246, the resin was washed as described in the legend to Fig. 5 and under “Materials and Methods,” and then aliquots were incubated for 1 h with wash buffer alone (W) or with buffer containing 0.5 m NaCl and 0.5% Triton X-100 (Tx), 100 or 300 μm YE-17 in 0.5 mNaCl/0.5% Triton X-100 (YE-17), or 10 mm EDTA (EDTA). After centrifugation, the supernatant (A) and pellet (B) were analyzed on Western blot with an antiserum to PC1. C, PC1 was bound to GST-proSAAS 221–246 and washed as described for A and B. Then the mixture was incubated for the indicated time (min) in buffer containing 0.5 m NaCl/0.5% Triton X-100 (Tx) and either 100 μm YE-17 or 100 μmpyroGlu-Arg-Thr-Lys-Arg-AMC substrate (Sub), the tubes were centrifuged, and the pellet was extracted and analyzed on Western blot for PC1 immunoreactivity. The relative positions of prestained molecular weight markers (Life Technologies, Inc.) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONOne of the major findings of the present study is that proSAAS is selective for PC1. Although relatively high concentrations of the C-terminal inhibitory peptide YE-17 do inhibit some of the other PCs, this could simply be competition with substrate and not tight binding as found for proSAAS and PC1. The low nm Ki for the C-terminal proSAAS peptides with PC1 is likely to be within the physiological range based on the relatively high abundance of proSAAS-derived peptides detected upon mass spectrometry of mouse brain (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar).Another important finding is that the inhibitory region of proSAAS is within a short 8–12-residue sequence near the C terminus. This region of proSAAS (VLGALLRVKRLE) is completely conserved among human, rat, and mouse (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar) and has sequence similarity (5 matches over 12 residues) to both the C-terminal region and the pro-region of rat PC1. Interestingly, both the pro-region (8Boudreault A. Gauthier D. Lazure C. J. Biol. Chem. 1998; 273: 31574-31580Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and the C-terminal region of PC1 (27Jutras I. Seidah N.G. Reudelhuber T.L. Brechler V. J. Biol. Chem. 1997; 272: 15184-15188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) have been shown to be inhibitory toward PC1. However, the inhibitory sequences within these regions have not been defined, and it is premature to speculate that the regions in PC1 with amino acid similarity to proSAAS are functional. Interestingly, this region of proSAAS contains the LLRVKR sequence previously identified from a screen of a peptide combinatorial library (28Apletalina E. Appel J. Lamango N.S. Houghten R.A. Lindberg I. J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The hexapeptide acetyl-LLRVKR-amide was the most potent inhibitor of PC1 found from this analysis (28Apletalina E. Appel J. Lamango N.S. Houghten R.A. Lindberg I. J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Consistent with our finding that LE-8 inhibits PC1 in a purely competitive fashion, the hexapeptide was also found to be a competitive fast-binding inhibitor (28Apletalina E. Appel J. Lamango N.S. Houghten R.A. Lindberg I. J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Thus, the additional N- and/or C-terminal residues in proSAAS and in YE-17 contribute to the slow tight binding inhibition of PC1.Surprisingly, the short proSAAS peptides are more potent than full-length proSAAS as PC1 inhibitors. This may be due to improper folding of the majority of GST-proSAAS fusion protein produced in bacteria. Although the short 8–16-residue inhibitory peptides are not likely to be present in neuroendocrine tissues, a 4-kDa peptide that includes this 16-residue region has been detected upon overexpression of proSAAS in AtT-20 cells (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). This 4-kDa C-terminal fragment is the major form of C-terminal immunoreactive peptide secreted from the proSAAS-expressing AtT-20 cells (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). In addition to the 4-kDa fragment, shorter C-terminal fragments were detected in the media of these AtT-20 cells, indicating that cleavage had occurred at the critical Lys-Arg site within the inhibitory region (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar).Another major finding of the present study is that proSAAS binds tightly to PC1. Although other investigators have not detected proSAAS during searches of proteins that bind to PC1, 2N. Seidah, unpublished results., 3I. Lindberg, personal communication.these studies used [35S]Met-labeled cell extracts and would therefore not have detected proSAAS because this protein lacks Met (except for the initiation methionine, which is cleaved off with the rest of the signal peptide). Also like 7B2 (11Hsi K.L. Seidah N.G. DeSerres G. Chretien M. FEBS Lett. 1982; 147: 261-266Crossref PubMed Scopus (117) Google Scholar), proSAAS stains extremely weakly with Coomassie Blue and silver staining procedures and has an extremely low extinction coefficient at 280 nm because of the low abundance of aromatic residues (16Fricker 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. Douglass J. J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar). Thus, proSAAS has presumably been undetected in previous studies because of the inadequacy of tools to visualize this protein.The tight binding and potent inhibition of PC1 by proSAAS raises the issue as to how this inhibition is regulated. A likely mechanism is cleavage at the Lys-Arg sequence within the inhibitory region; as mentioned above, forms of smaller proSAAS peptides resulting from cleavage at this site have been detected in mouse brain and proSAAS-expressing AtT-20 cells (16Fricker L" @default.
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