Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing

Abstract

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 IC(50) of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.

Fig. 1.

Top, Nucleotide and deduced amino acid sequence of mouse proSAAS. The amino acid sequences of the peptides found to be elevated in tissues fromCpe^fat/Cpe^fatmice are underlined (double underlineindicates those peptides found in both a short and long form).Bottom, Amino acid identity among human, rat, and mouse proSAAS. Asterisks below the sequence denote residues conserved in all three species. Open arrowhead, Signal peptide cleavage site. Double arrows, Paired basic cleavage sites (RR, KR) that are used in theCpe^fat/Cpe^fatmouse. Single arrows, KxxR cleavage site used in theCpe^fat/Cpe^fatmouse. Half arrows, Additional predicted cleavage sites. The sequences of the peptides that correspond to those found in theCpe^fat/Cpe^fatmice (but without the C-terminal basic residues that would be removed by active CPE) are indicated by lines.

Fig. 2.

Northern blot analysis of proSAAS mRNA in human tissues. Northern blots containing poly(A) RNA (Clontech) were probed with ³²P-labeled human proSAAS cDNA, as described in Materials and Methods, and were exposed to x-ray film for 2 hr at −80°C.

Fig. 3.

In situ hybridization analysis of proSAAS mRNA in rat brain (A–D), adrenal gland (E, F), and spinal cord (G, H). For the adrenal and spinal cord, the left panels (E, G) show autoradiograms from x-ray film, and the right panels (F, H) show emulsion-dipped sections. AH, Anterior hypothalamus; AM, adrenal medulla; ARC, arcuate nucleus;BNST, bed nucleus of the stria terminalus;CTX, adrenal cortex; DH, dorsal horn;DMH, dorsal medial hypothalamus; G, glia;MPO, medial preoptic area; N, neuron;Pir, piriform cortex; SNc, substantia nigra pars compacta; SO, supraoptic nucleus;VH, ventral horn; VMH, ventral medial hypothalamus.

Fig. 4.

Expression of rat proSAAS in AtT-20 cells.A, Western blot analysis of expression of proSAAS in AtT-20 cells. Left, Wild-type (wt) cells or cells transfected with proSAAS cDNA (SAAS) in the pcDNA3 vector were analyzed on a Western blot and probed with an antiserum directed against proSAAS 245–260. Right,Media were collected from proSAAS-expressing cells treated for 30 min with control medium (C), or with media containing 10 μm forskolin (F) or 5 mm 8-Br-cAMP (A). The media were then analyzed on a Western blot with antiserum to proSAAS 245–260. The position and molecular mass (in kilodaltons) of prestained molecular weight markers (Life Technologies) are indicated. B,Confocal microscopy of immunoreactive SAAS in AtT-20 cells overexpressing proSAAS. Cells were cultured, fixed, permeabilized with 0.1% Triton X-100, and stained with rabbit antiserum to proSAAS 245–260, as described in Materials and Methods. Bound antiserum was detected with fluorescein-labeled anti-rabbit antiserum. Three proSAAS-expressing cell lines were examined with similar results. Scale bar, 10 μm.

Fig. 5.

A, Pulse/chase analysis of proSAAS processing in AtT-20 cells overexpressing rat proSAAS. Cells were labeled with [³H]Leu for 20 min and then chased for 0, 15, 30, or 90 min in unlabeled Leu, and then the media and cell extracts were immunoprecipitated with antisera to the C-terminal region (245–260) or the N-terminal region (42–59) of pro- SAAS. The immunoprecipitates were fractionated on a denaturing polyacrylamide gel that was treated with Fluro-hance (Research Products International) and exposed to film for 3 d. The position and molecular mass (in kilodaltons) of prestained molecular weight markers (Life Technologies) are indicated. B,Analysis of media from wild-type AtT-20 cells (bottom) and rat proSAAS overexpressing cells (top) using matrix-assisted laser desorption ionization time of flight mass spectrometry. Predicted POMC- and proSAAS-derived peptides that are within 1 mass unit of the observed peaks are indicated.LPH, lipotropin; ACTH, adrenocorticotropic hormone; CLIP, corticotropin-like immunoreactive peptide.

Fig. 6.

Pulse/chase analysis of proopiomelanocortin processing in wild-type AtT-20 cells (wt) and in AtT-20 cells overexpressing proSAAS (SAAS). Cells were labeled with ³⁵S-Met for 10 min and then chased for 30 or 90 min in unlabeled Met, and then the media were immunoprecipitated with antisera to ACTH (left) or β-endorphin (right).Top, Exposure of the gel to x-ray film for 14 hr.Bottom, Exposure for 4 d.

Fig. 7.

Inhibition of PC1 by proSAAS. A,Purified GST-proSAAS or GST at the indicated concentrations was combined with PC1 or PC2 and 100 μm RTKR-AMC substrate, as described in Materials and Methods. The results represent mean ± SEM from three determinations. The experiment was performed with two independent preparations of GST and GST-proSAAS, with similar results.B, Purified GST-proSAAS or GST at the indicated concentrations were combined with PC1 and various concentrations of RTKR-AMC substrate, as described in Materials and Methods. Kinetic constants were determined from linear regression analysis of plots of the inverse of the reaction velocity (with GST-proSAAS or GST) versus the inverse of the substrate concentration (Lineweaver–Burk plot). The experiment was performed with two independent preparations of GST-proSAAS and GST with similar results.