Peptides Derived by Processing of Rat Prosomatostatin near the Amino-Terminus: Characterization, Tissue Distribution, and Release*

Abstract

Mammalian prosomatostatin (pro-S) undergoes extensive processing at the C-terminal segment where the somatostatin-like biological activities (S-14 and S-28) reside. The recent discovery of pro-S-(1-10) (antrin) as a prominent mature product in the stomach suggests that pro-S may also be processed at the N-terminus. In the present study we have developed an antibody directed against the N-terminal segment of pro-S-(1-10) capable of detecting peptides extended at the C-terminus of pro-S-(1-10) to characterize N-terminal processing of rat pro-S. Specifically, we have 1) examined the relative abundance of pro-S-(1-10)-like immunoactivity [pro-S-(1-10)] in different somatostatin tissues as an index of tissue-specific N-terminal processing, 2) compared the concentrations of pro-S N- and C-terminal immunoreactive peptides, 3) used HPLC and region-specific RIAs directed against both the N- and C-terminal segments of pro-S to identify and characterize novel N-terminal peptides, 4) studied the tissue distribution and release of the N-terminal peptides; and 5) characterized and quantified a 7-kDa molecule equivalent to pro-S without the C-terminal S-28 sequence. Acetic acid (1 M)-pepstatin extracts of hypothalamus, cerebral cortex, antrum, jejunal mucosa, and pancreas were fractionated by reverse phase and gel permeation HPLCs. Whole tissue extracts as well as the column effluent were monitored by region-specific RIAs using antibodies against pro-S-(1-10), S-28-(1-12), and S-14. Other than the pancreas, all S-producing tissues were rich in pros-S-(1-10) LI. Its concentration was 1- to 4-fold lower than those of S-14 LI and S-28-(1-12) LI. Tissue pro-S-(1-10) LI was hetergeneous, consisting of at least eight molecular forms with respective mol wt of 1,00 (1 kDa), 1,500 (1.5 kDa), 2,500 (2.5 kDa), 3,500 (3.5 kDa), 4,500 (4.5 kDa), 7,000 (7 kDA), 8,000 (8 kDa), and 10,000 (10 kDa). Based on the simultaneous presence or absence of C-terminal immunoreactivity, the 10-kDa form corresponded to pro-S, 8 kDa to pro-S-(1-76), and 7 kDa to pro-S without the S-28 sequence. The predominant N-terminal forms corresponded to 1 kDa [pro-S-(1-10)] and 7 kDa. The 1-, 1.5-, 2.5-, and 7-kDa forms were identified as secretion products in portal blood or in medium from cultured 1027 B2 islet somatostatin cells. These data suggest that pro-S undergoes extensive N-terminal processing to generate not only antrin, but also a number of other mature products which remain to be fully characterized. Antrin occurs in all S-producing tissues and is not confined solely to the antrum. The finding of high concentrations of 7 kDa in tissues that synthesize predominantly S-14 (antrum) as well as in S-28-producing tissues (jejunum and brain) suggests that this peptide is derived through both direct processing of pro-S.fwdarw.S-28 + 7 kDa as well as through sequential breakdown of pros-S .fwdarw.S-14 + pros-S-(1-76) .fwdarw. 7 kDa + S-28-(1-12).