Peptides,Vol. 12, pp. 601-607. ¢ PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Characterization of Large Neuromedin-N Using Antisera Towards Regions of the Neurotensin/Neuromedin-N Precursor R O B E R T E. C A R R A W A Y , S A N K A R P. M I T R A A N D C A R O L P A R A D I S E

Department of Physiology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655 Received 21 N o v e m b e r 1990

CARRAWAY, R. E., S. P. MITRA AND C. PARADISE. Characterization of large neuromedin-Nusing antisera towards regions of the neurotensin/neuromedin-Nprecursor, PEPTIDES 12(3) 6ol--607, 1991.--Processing of the precursor to neurotensin/ neuromedin-N was studied in brain and intestine from four mammalian species (dog, cat, guinea pig and rat) using previously characterized immunoassays for neurotensin and neuromedin-N, as well as newly developed assays towards the 35-44 sequence (P1) and the 70-85 sequence (P2) of the canine precursor. While neurotensin was the major product (--98%) with neurotensin immunoreactivity in brain and ileum, a large molecular form of neuromedin-N was found to comprise 55-91% of the neuromedin-N activity in the ileum of these species and only 2-8% that in brain. Large neuromedin-N, which behaved as a single substance during multiple chromatographic steps, was found to cross-react in the assays for P1 and P2, indicating that this molecule extended at least from residues 40-148, neuromedin-N being located at its C-terminus. Western blots confLrmed the results obtained by irnmunoassay. Partially purified preparations of large neuromedin-N from dog, cat and rat were also found to contract the isolated guinea pig ileum, exhibiting potencies near to that of neuromedin-N. These results indicate that tissue-specific storage of large neuromedin-N, a biologically active molecule with > 100 amino acids, occurs in these four mammals. Neuromedin N

Neurotensin/neuromedin-N precursor

ABBREVIATIONS

NEUROMEDIN-N (NMN) and neurotensin (NT), putative regulatory peptides found in the central nervous system and gastrointestinal tract (6, 8, 21), are apparently excised from a 170 amino acid precursor, the sequence of which was derived from the corresponding cloned canine cDNA nucleotide sequence (11). Work with proteins extracted from a human neuroendocrine pancreatic carcinoma has now confirmed the cDNA-based prediction of a l : l molar ratio of NT to NMN within a precursor of - 18 kilodaltons (9). Early reports concerning differential expression of the avian and reptilian counterparts to these two peptides in chicken brain and gut (10), in turtle retina (14) and in certain neurons of the pigeon, turtle and hamster globus pallidus (24) suggested that tissue-specific processing of this precursor might occur. Additional studies in cat established that the pattern of distribution for immunoreactive NMN (iNMN) differed from that of immunoreactive NT (iNT), such that the ratio of these two activities varied >10-fold depending on the tissue examined (8). Recently, we demonstrated that extracts of canine small intestine contained primarily large molecular forms of NMN, whereas the predominant species in brain was NMN itself (7). NT, on the other hand, was found only as the small peptide. The goals of the studies reported here were to determine whether this difference in the form of NMN within brain and intestine was a general phenomenon for mammals and to perform an immunochemical and biological characterization of large NMN.

NMN, neuromedin-N; iNMN, immunoreactive NMN; NT, neurotensin; iNT, immunoreactive NT; P1, EADLLTNMHTSKI; P2, EETGEFREEELIT; P3, QQWELIQEDVL; P4, DAGNDKNEKEEVI; HPLC, high pressure liquid chromatography; RIA, radioimmunoassay; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis. METHOD

Peptide Synthesis Solid phase peptide synthesis using Boc-amino acids and HF cleavage was performed in the Peptide Core Facility at UMMC with a model 430A Applied Biosystems Peptide Synthesizer and PAM resin (0.5 g, ~ 0 . 6 retool/g). The completed peptides, [Tyr°]prepro-NT/NMN(33--45), [Tyr°]prepro-NT/NMN(72-84), [Tyr°]prepro-NT/NMN(117-127) and [Tyr°]pre[~ro-NT/NMN(128140), [Tyr°]P1, [Tyr°]P2, [Tyr°]P3 and [Tyr"]P4, respectively, were cleaved from the resins using HF, washed using diethyl ether and purified by chromatography on Sephadex G-25 (column size, 5 x 120 cm) using 5% acetic acid. Analytical HPLC on ~x-Bondapak C18 ( 3 . 9 x 3 0 0 ram) using a linear gradient (60 min) from 0.1% trifluoroacetic acid to 60% acetonitrile indicated that a single component accounted for >90% of the absorbance at 280 mix. The extended forms of NMN, peptide K-22 (EEVIKRKIPYIL) and peptide R-12 (KIPYILKRQLYENKP-arnide)

601

602 were synthesized using the solid phase method with Fmoc-amino acids, Wang resin (0.1 mmol amino acid), and TFA cleavage. After cleavage from the resin, the completed peptides were washed with ether and ethyl acetate and purified by HPLC on a column (19 x 300 mm) of ix-Bondapak C18 using a linear gradient (80 min) from 0.1% TFA made 15% acetonitrile to this buffer made 60% acetonitrile with a flow rate of 6 ml/min. Amino acid analyses on acid hydrolysates gave near integral molar ratios for the expected amino acids.

Preparation of Antisera [Tyr°]P1 (12 mg) was coupled to succinylated thyroglobulin (6 mg) as previously described for NT (4). [Tyr°]P2 (10 rag) was coupled to bovine thyroglobulin (10 mg) using bis-diazotized benzidine as described for TRH (1). The coupling yields were ~45% and ~65% for P1 and P2 based on the amounts of |25I-labeled peptide incorporated into the conjugates. Each conjugate was emulsified with complete Freund's adjuvant and administered intradermally to two New Zealand White rabbits ( - 2 mg/animal) as previously described (3). The rabbits were boosted 4 months later with 0.2 mg conjugate and then bled bimonthly.

lodination and RIA Iodination of all peptides was carried out with Na~25I (New England Nuclear, Boston, MA; 1 mCi) and chloramine T (Fisher Scientific, Pittsburgh, PA; 25 Ixg) as previously described for NMN (8). The iodinated peptides were purified by HPLC on ~-Bondapak C18 as described (8), yielding tracers with near maximal specific activities (1000-2500 cprn/fmol) which were stable for up to 3 months when stored at -20°C in PBS made 10 mg/ml in bovine serum albumin and 20% ethanol. The RIA procedures for NT and NMN were as previously described (5,8). Similar methods were employed for peptides P1 and P2. Samples and standards were incubated with tracer (20,000 cpm) and diluted antiserum under conditions permitting - 2 5 % maximal binding to occur. After equilibrium was attained at 4°C (18-24 h), bound and free radioactivity were separated using a charcoal-dextran mixture (5). Bound radioactivity was determined using a 16-well ~/-counter (Nuclear Enterprises, Edinburgh, Scotland) with on-line computerized data analysis using a log-logit linearization program (IN/US Service Corp., Fairfield, NJ). Intraassay and interassay coefficients of variation were 9.2% and 12.5% for the P1 assay and 13.9% and 21.0% for the P2 assay, respectively (n = 5).

Extraction of Tissues Tissues were extracted as described previously (7). In brief, tissues were excised from anesthetized animals (Nembutal for dogs, cats and rats; halothane for guinea pigs) and placed on ice; animals were then euthanized. Tissues were minced and extracted with 10 volumes (v/w) of ice-cold 0.1 N HC1 containing 15 Ixg/ml pepstatin A (Sigma Chemical Co., St. Louis, MO) and the homogenates were placed in a boiling water bath for 15 min and then centrifuged at 12,000 x g for 15 min. Supernatants were collected and stored at -20°C for < 1 week before chromatography was performed.

CARRAWAY, MITRA AND PARADISE

tic acid. After the sample (6 ml) was applied, 100 ml eluate was discarded and fractions of 8 ml were collected. Recovery of immunoreactivity was >80%. Reverse-phase high pressure liquid chromatography (HPLC) employed a column of Ix-Bondapak C18 (0.39x 30 cm) equilibrated with 0.1% trifluoroacetic acid. After injection of the sample, a linear gradient (5 min) was applied to 15% acetonitrile, followed by a linear gradient (30 min) to 75% acetonitrile at 1.5 ml/min. Recovery of immunoreactivity was >90%. Electrophoresis was performed in 12 cm polyacrylamide gels using 5% acetic acid containing 2.5 M urea as described previously (7). Recoveries were - 2 0 % . Histone standards were purchased from Sigma Chemical Co. (St. Louis, MO).

Western Blot Partially purified canine large NMN (post-Sephadex G-75, 1.5 nmol iNMN), reduced with 1% mercaptoethanol, 4% SDS in 0.1 M Tris-HC1, pH 6.8, was subjected to SDS-gel electrophoresis in 6 lanes of a 20% polyacrylamide slab gel according to Laemmeli (20). Protein standards from LKB Pharmacia (Piscataway, NJ) with molecular weights of 16.9, 14.4, 8.2, 6.2, and 2.5 occupied one lane and were located by Coomassie staining. The proteins in samples of large NMN were electrophoretically transferred to nitrocellulose paper (Bio-Rad Transblot medium) using a Hoefer apparatus with 1 amp for 4 h at 4°C in 0.19 M glycine, 0.02 M Tris-HC1 (pH 8.3) with 20% methanol. The paper was washed with Tris-buffered saline (TBS, 20 mM Tris-HC1, pH 7.5, 0.5 M NaC1), incubated 16 h with 3% nonfat dry milk in TBS at 4°C, washed with TBS and cut into 3 strips. Incubation with primary antibody was done at 1:100 dilution in 1% milk for 48 h (1 strip each for anti-NMN, anti-P1 and anti-P2). The strips were washed exhaustively with TBS made 0.1% Tween-20 and incubation with biotinylated secondary antibody for 2 h as recommended by Vectastain elite kit was performed (Vector Labs, Burlingame, CA). After exhaustive washing, the strips were stained using ABC reagents and 0.1% diaminobenzidine with 0.01% H202.

Bioassay The contractility of freshly dissected guinea pig ileum was examined using a 10 ml organ bath perfused with Tyrode's solution maintained at 37°C and aerated with a mixture of O2-CO 2 (95:5). The animals were anesthetized with halothane and the tissues immediately placed into oxygenated Tyrode's. Tissue sections ( - 2 cm) were suspended in the bath and isotonic contractions were recorded under a tension of - 1 g using a Grass Polygraph and tension transducer. Mounted tissue was allowed to equilibrate for 1 hour before testing. The ECho was defined as the concentration which produced half of the maximal contraction by each peptide tested. Samples were applied at 10-min intervals with multiple washes in between to avoid desensitization. Dose-response curves for NT and NMN were performed for each tissue at the start and end of each experiment. Large NMN (post-HPLC), dissolved in 0.85% saline containing 10 mM acetic acid, was diluted at least 20-fold during testing. This buffer did not contract the tissue nor affect the response to NMN. RESULTS

Chromatography and Electrophoresis

Antisera

Gel permeation chromatography was performed using a column of Sephadex G-75 (2.5 x 95 cm) equilibrated with 5% ace-

Antisera were raised towards the partial sequences, P1 and P2, of the mRNA-predicted NT/NMN precursor as shown in

LARGE NEUROMEDIN N

33 25 [ 45

1

SIGNAL

P1

603

72 P2

V///A-R

Radioimmunoassays

148 163 143 \151 I 170

84

In general, the antibodies obtained towards the regions of the precursor were of lower affinity than those obtained towards NT and NMN. However, each RIA was shown to be specific in that cross-reactions with other partial sequences of the precursor and other commercially available peptides were insignificant (Table 1).

NMN NT

Z

Concentrations of Immunoreactivity in Tissues When 0.1 N HCI extracts of canine brain and ileum were assayed for the various immunoreactivities, the results in Table 2 were obtained. Concentrations of all activities were higher in ileum than in brain ( ~ 100-fold) and while the amounts of iNT, iNMN and iP2 were similar in each tissue, the level of iP1 was 6--8-fold lower.

FIG. 1. Diagrammatic representation of the 170 residue canine NT/ NMN precursor and location of peptide sequences, P1, P2, NMN and NT. Numbers denote residue number at beginning and end of indicated peptide according to mRNA sequence by I~bner et al. (11). NT and NMN are bordered by double basic residues. The amino acid sequences of Pl and P2 are shown.

Gel Chromatography of Tissue Extracts As shown previously (7), >75% of the NMN-related activity present in an acid extract of canine ileum eluted as a large molecular form (Mr, 15-20 kDa) when subjected to gel chromatography on Sephadex G-75 (Fig. 2A). Similar proteins could also be demonstrated in extracts of ileum from cat (Fig. 2B), guinea pig (Fig. 2C) and rat (Fig. 2D). While large NMN accounted for 53-85% of total iNMN, only ~ 2 % of iNT eluted as a large molecular form, ~98% eluting in the region of NT itself (Fig. 2A-D; see legend for statistics). When extracts of brain were

Fig. 1. Other antisera, previously raised in our laboratory towards the biologically active peptides, NT and NMN, were also available for the study. Radioimmunoassays towards these peptides were developed using ~25I-labeled tracers as described in the Method section, and their characteristics are summarized in Table 1.

10 A -

DOG

A2V*

hit NMN

E-

120

DOG

30

75 "/.CHnCN

,fit..q~ . 10

B - CAT

]

p,

NMN

60

20

80

_.

Z'." "-,. .

Z 'TO

25 ~

i

:--

CAT INMN~

A

Z •

z

/

2

8

-OUINEAPIG

',

t10 ,-E

,if % / - -

"" E

40

]' ----~ =i 10 d -

GUINEA PIG

1 INMN

INM

1

,' ,'

"o. o ~

"4~ 0,4

D-

2

5

| -~2

8

~1 '

/

RAT

H-

,

,

,,J, ~

,

RAT

INMN ~]

10

30 FRACTION NUMBER (8 rot)

,50

..¢.r 1()

'

2'0

:30

RETENTION TIME (mln)

FIG. 2. Left panel: Gel chromatography of acidic extracts of ileum from dog (A), cat (B), guinea pig (C) and rat (D) on Sephadex G-75 and profile of iNMN and iNT. Multiples of the void volume are indicated by Vo and the elution positions for synthetic NT and NMN are marked by arrows. Large NMN, defined as the peak eluting just before 2 Vo, accounted for 91 - 3% (dog), 73 ---7% (cat), 84+__9% (guinea pig) and 55__.8% (rat) of total iNMN (mean___SEM, n=3). Right panel: Chromatography of large NMN from (A-D) on I~-Bondapak C18 and measurement of iNMN activity: dog (E), cat (F), guinea pig (G), and rat (H). The dashed line shows the gradient of acetonitrile used and the arrow indicates the elution position of synthetic NMN.

604

CARRAWAY, MITRA AND PARADISE

TABLE 1 PROPERTIES OF THE ANTISERA TOWARDS P1, P2, NMN AND NT

Sequence

Antiserum Titer

ICs0* (pmol)

Sensitivity* (pmol)

P1

EADLLTNMHTSKI

1:2500

1.5

0.10

P2

EETGEFREEELIT

1:25,000

0.5

0.06

NMN

KIPYIL

1:25,000

0.3

0.03

NT

pQLYENKPRRPYIL

1:200,000

0.02

0.002

Peptide

% CrossReactiont [Tyr°]P1, 100% All others,

neuromedin-N precursor.

Processing of the precursor to neurotensin/neuromedin-N was studied in brain and intestine from four mammalian species (dog, cat, guinea pig and rat) ...
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