Differential Processing of Neurotensin/Neuromedin N Precursor(s) in Canine Brain and Intestine*

By using a radioimmunoassay for neuromedin N (NMN), a hexapeptide in the neurotensin (NT) family, extracts of canine small intestine were found to contain primarily (greater than 75%) large molecular form(s) of NMN, whereas the predominant species in brain was NMN itself. Large NMN was highly basic (pI greater than 9) and during sodium dodecyl sulfate gel electrophoresis gave two components of approximately 17 kDa (75%) and approximately 8 kDa (25%). Large NMN, like NT, was localized primarily to the mucosal layer of the jejunoileum. It was also present in highly purified (25% pure) mucosal N-cells, where it appeared to be concentrated within dense secretory vesicles. The amino acid sequence of a 21-amino acid fragment cleaved from the C-terminal region of large NMN was identical to residues 128-148 of the canine NT/NMN precursor predicted from cDNA work. These results suggest that tissue-specific processing of the NT/NMN precursor occurs in the dog, giving rise to NMN in brain and large NMN in small intestine.

By using a radioimmunoassay for neuromedin N (NMN), a hexapeptide in the neurotensin (NT) family, extracts of canine small intestine were found to contain primarily (>75%) large molecular form(s) of NMN, whereas the predominant species in brain was NMN itself. Large NMN was highly basic (pI>9) and during sodium dodecyl sulfate gel electrophoresis gave two components of -17 kDa (75%) and -8 kDa (25%). Large NMN, like NT, was localized primarily to the mucosal layer of the jejunoileum. It was also present in highly purified (25% pure) mucosal N-cells, where it appeared to be concentrated within dense secretory vesicles.
The amino acid sequence of a al-amino acid fragment cleaved from the C-terminal region of large NMN was identical to residues 128-148 of the canine NT/NMN precursor predicted from cDNA work. These results suggest that tissue-specific processing of the NT/NMN precursor occurs in the dog, giving rise to NMN in brain and large NMN in small intestine.

Neuromedin
N (NMN)' and neurotensin (NT), peptides which share structural (1, 2) and biological (3) properties and which are distributed similarly in the cat (4), are present in a 1:l molar ratio within a 170-amino acid precursor, the sequence of which was predicted from cDNA work on mRNA obtained from canine intestinal N-cells (5). Recent work* has shown, however, that the ratio of NT to NMN in extracts of canine intestine as well as in partially purified N-cells is -7, suggesting that other peptides containing the NMN sequence may exist.
Here we present evidence indicating that the major product of precursor processing for NMN in canine intestine is an Nterminally extended, large molecular form. Furthermore, the quantity of this peptide present in the tissue accounts for the discrepancy in the NT:NMN ratio. In addition, we show that very different results are obtained with canine brain, which exhibits an NT:NMN ratio near 1.0.
* This work was supported by National Institutes of Health Grant DK28565. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. . For canine jejunoileum, however, far lower amounts of iNMN were obtained with acid/acetone than with HCl, whereas the yield of iNT was similar with these two procedures (Table I; iNT:iNMN = 1.5 and 5.0, respectively. Since added synthetic NMN was retrieved from these extracts in similar amounts (yields: acid/ acetone, 77 f 9%, and HCI, 86 + 8%; n = 3), these results suggested that the majority of iNMN in the jejunoileum (-80% of total) was due to another peptide which was insoluble in acetone.
Chromatography-Indeed, when HCl extracts of the jejunoileum were subjected to gel chromatography on Sephadex G-75, 70-90% of iNMN was found to elute as larger substances than synthetic NMN, whereas >95% of iNT behaved as synthetic NT (Fig. lA). In this chromatogram, the major substance (peak 2) comprised -76% of total iNMN, whereas NMN (peak 3) accounted for only -12%. Upon rechromatography, the elution behavior of the material in peak 2 was unaffected by treatment with 6 M guanidine HCl at 80 "C (data not shown).
Brain extracts, on the other hand, gave profiles of iNMN and iNT indicating the presence of <5% large molecular forms (Fig. 1B).
Electrophoresis of Large iNMN-When the material in peak 2 ( Fig. lA) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 7.5 and 15% gels, two major proteins were identified (Fig. 2): a 17-kDa species (-75% of total) and an 8-kDa species (-25% of total). Electrophoresis on acid/urea gels indicated that these proteins were highly basic, migrating as far as histones H3 and H4 (Fig. 4). Isoelectric points for these proteins were between 8.5 and 9.5 (data not shown).
Tissue and Cellular Distribution-When dissected regions of the jejunoileum were analyzed for iNMN and iNT, these activities were found to be similarly distributed (Table I). For iNMN, -97% was localized to the mucosa, -2% was found in the submucosa, and -1% was present in the muscle.
Highly purified (-25% pure) N-cells, dispersed from jejunoileal mucosa and purified using Percoll, elutriation, and selective culturing, also contained both iNMN and iNT in similar proportions (  chromatographed on Sephadex G-75, gave primarily the large molecular form of iNMN (65-70%) along with NMN itself (30-35% (results not shown); n = 2). In addition, the same immunoreactive protein was observed during electrophoresis in acid/urea gels as was obtained from whole jejunoileum (Fig.   4B).

Subcellular
Distribution-During equilibrium sucrose density centrifugation of an isotonic homogenate of dispersed mucosal cells, iNMN and iNT banded together at a density of -1.19 (Fig. 5), which is near the density previously determined for feline N-cell vesicles (4).

Partial
Sequence of Large iNMN-The C-terminal specificity of the NMN antiserum (4) suggested that the NMN-like moiety was located at the C terminus of large NMN. This was substantiated by the fact that carboxypeptidase treatment destroyed the immunoreactivity (95% reduction), whereas treatment with aminopeptidase M had no effect (~5% reduction) .
A brief treatment with pepsin (0.1 mg/ml, 60 min, 40 "C) fragmented the molecule without any loss of immunoreactivity, and the presumed C-terminal portion containing the NMN-like moiety was then purified. Using the procedure described in Table II, a Pl-amino acid peptide was isolated (yield, -19%; purification, lOO,OOO-fold). The final step gave a single peak of optical density coincident with that for iNMN activity (Fig. 7) The isolated peptide cross-reacted -65% in the radioimmunoassay for NMN. Its amino acid composition (Table III) was consistent with the sequence determined by automated Edman degradation (Table IV). This sequence was identical to that predicted by cDNA studies for the 21 residues immediately N-terminal to and including NMN in the NT/ NMN precursor (Fig. 3).

DISCUSSION
The major finding reported here is that an N-terminally extended form of NMN is the predominant species present in extracts of canine intestine (>75% of total iNMN), whereas NMN itself is the primary product in canine brain (>95% of total iNMN).
The isolation of this large NMN has not been accomplished yet; however, we have been able to estimate the size of the major component (-17 kDa) and to investigate the location of the NMN moiety which appears to be at the C terminus. In addition, the amino acid sequence of a 21-amino acid fragment cleaved from the C-terminal region of large iNMN has been determined.
The identity of this sequence to residues 128-148 of the NT/NMN precursor predicted from cDNA studies (5) suggests that large NMN originates from the same precursor molecule as NT and NMN. Although there is no evidence as yet for gene duplication events or alternate gene splicing for the NT/NMN precursor, the possible existence of a distinct precursor for large NMN must be considered. However, given the 21-amino acid identity with the NT/NMN precursor, the simplest hypothesis at this point is that tissue-specific processing of the same protein gives rise to NMN in brain and large NMN in intestine.
Although we were able to estimate the molecular size of large NMN and to sequence a 21-amino acid fragment of the molecule, its precise alignment with the NT/NMN precursor is not yet clear. The l7-kDa size is near that predicted (-15 kDa) for the molecule extending from the end of the putative signal sequence (residue 25) to the end of NMN (residue 148). However, the very basic nature of the intestinal peptide (p1 >9) is difficult to reconcile with the rather acidic character of this segment of the precursor (p1 <7). A slightly shorter sequence (residues 37-158) would have 6 fewer acidic residues and a basic p1. Since there are several potential glycosylation sites within the precursor, it is also possible that both the charge and the size of large NMN are functions of a carbohydrate portion.
Work presented here concerning the cellular distribution of large NMN and iNT in canine intestine strongly suggests that large NMN coexists with iNT in mucosal N-cells. Thus, large NMN (like NT) was localized to the mucosal region (-97%) and found to be highly concentrated within partially purified (-25% pure) intestinal N-cells. Furthermore, these two peptides co-migrated during sucrose gradient centrifugation of N-cell homogenates, indicating their association with dense secretory-like vesicles. The fact that the ratio of iNT to iNMN was constant and nearly 1 for these various preparations (Table I) is consistent with the idea that large NMN and NT are co-stored within N-cells. Immunohistochemical studies to address this issue further are currently underway. Previous work on birds and reptiles by Reinecke (7) indicates that intestinal cells staining for LANT-6, the presumed avian counterpart to NMN (8,9), also stain for NT.
The biological significance of large NMN is not yet known. Preliminary studies4 in our laboratory indicate the presence of large molecular form(s) of iNMN in canine blood at concentrations which appear to exceed those of NT. These peptides may be secreted forms of intestinal iNMN; and if they can exert biologic actions themselves or liberate NMN upon proteolytic cleavage, they could be physiologically significant. The greater stability generally displayed by large molecular forms as opposed to smaller forms of biologically active peptides in the circulation (e.g. CCK-58 and CCK-33 uersus CCK-8 (10, 11)) makes them attractive candidates as hormones.