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ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.

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 Cpe(fat)/Cpe(fat) 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 Cpe(fat)/Cpe(fat) 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 Cpe(fat)/Cpe(fat) 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 Cpe(fat)/Cpe(fat) 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.

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 microm 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 apparent K(i) 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 glutathione S-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 m NaCl. The removal of Ca(2+) 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.

In vitro assays have demonstrated that peptides derived from the recently-identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre- and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post-mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid-gestation with marked accumulation of two C-terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Members of several metalloprotease families have been proposed to be involved in non-classical processing of neuroendocrine precursors. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits a neuroendocrine distribution, intracellular subcellular localization, and an acidic pH optimum. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. These results are consistent with an important role for ECE-2 in the processing of regulatory peptides at non-classical sites.

ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse–chase analysis using [3H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60–80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

Cpe fat /Cpe fat mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe fat /Cpe fat mice. These “orphan” peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems.

Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. 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. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC50of 590 nmbut does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.