Bioehimica et Biophysiea Aeta, 493 (1977) 452-459 © Elsevier/North-Holland Biomedical Press

BBA 37727

SEQUENCE ANALYSIS OF PORCINE GUT GLI-1

HENNING JACOBSEN, ANNI DEMANDT, AL1STER J. MOODY and FINN SUNDBY Novo Research Institute, Copenhagen (Denmark)

(Received March 4th, 1977)

SUMMARY

A protein from porcine gut with 100 amino acid residues (porcine gut GLI-1) and having glucagon-like immunoreactivity has been characterized by partial se- quences. The sequence of the C-terminal amino acid residues is -Met-Asn-Thr-Lys- Arg-Asn-Lys-Asn-Asn-Ile-Ala and includes the C-terminal amino acid residue sequence (-Met-Asn-Thr) of porcine glucagon. Evidence is presented that the glucagon sequence -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- is found in the gut GLI-1 as well. The data support the theory that gut GLI-I contains the full glucagon sequence and that gut GLI-1 and glucagon are formed from a common precursor.

INTRODUCTION

Extracts of porcine gut contain several polypeptides which have an immuno- determinant similar to that in the region 2-23 of glucagon [1, 2], but do not have a detectable immunodeterminant similar to that of glucagon (24-29). These polypep- tides are known as gut GLI's, the GLI standing for Glucagon Like lmmunoreactivity. Gut GLI's are synthetised in endocrine cells in the intestinal mucosa [3] and are released from the intestine by the ingestion of foodstuffs, in particular carbohydrates [4]. It is of clinical importance to know whether the immunochemical similarity between gut GLI's and glucagon is the consequence of gut GLI's having an extensive structural and biological resemblance to glucagon. Our approach to this question has been to isolate and characterise porcine gut GLI's.

The first pure porcine gut polypeptide (gut GLI-1) having glucagon-like immunoreactivity has been isolated, and its amino acid composition and the sequence of the 13 N-terminal amino acid residues and of the 2 C-terminal amino acid residues were determined [5]. Glucagon may be present as a part of the gut GLI-1, judging from the amino acid composition, but the published N- and C-terminal sequences differed from those of glucagon. The C-terminal amino acid sequence was -Ile-Ala, which is identical with the C-terminal residues of a glucagon-containing peptide isolated from commercial glucagon by Tager and Steiner [6]. The present paper reports the isolation and sequence determination of a C-terminal decapeptide from

Abbreviation: dansyl, 5-sulphonyl-l-dimethylaminonaphthalene.

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gut GLI-1 and shows its marked similarity with the C-terminus of the proposed proglucagon fragment of Tager and Steiner.

Furthermore, autoradiograms of peptide maps of chymotryptic digests of radiolabeiled glucagon and of radiolabelled gut GLI-1 suggest the presence of two similar peptides in both materials.

MATERIALS

Porcine gut GLI-1 was purified as reported previously [5]. The following auxiliary polypeptide and enzymes were used: porcine glucagon (NOVO Lot No. G 425043), a-chymotrypsin (NOVO), Armillarea mellea protease (ICI Pharmaceuticals Division), and carboxypeptidase Y purified from yeast by affinity chromatography [7].

Micropolyamide plates, F 1700, Schleicher & Schfill, Dassel; carrier-free solution of 125I-, code No. IMS 30, Radiochemical Centre, Amersham; Shandon thin-layer electrophoresis apparatus Mk II; cellulose thin-layer plates, Avicel 250 #m, 20 × 20 cm, Anachem Ltd., Luton, purified before use by ascending chromatography in 5 ~o pyridine and 1.5 ~ formic acid, successively [8].

Pyridine, phenylisothiocyanate and hydrochloric acid for hydrolysis of pep- tides were redistilled and stored under nitrogen at --20 °C. Other reagents were of analytical grade.

METHODS

Treatment with cyanogen bromide 215 nmol of gut GLI-1 (2.5 mg) was treated with cyanogen bromide (185 molar

excess over methionine) in 200/tl 70 ~o formic acid at room temperature for 44 h. After repeated lyophylization, the peptide mixture was dissolved in 50 ~ acetic acid and the reaction products separated by gel filtration on Sephadex G 50, fine (Phar- macia). Specifications as described in Fig. 1. Peptides that did not absorb at 280 nm were detected by taking out aliquots of 1 ~o of fraction size, drying down, and treating with dansyl chloride [9]. After hydrolysis the N-terminal amino acid residues were identified as dansyl amino acids by thin-layer chromatography on polyamide sheets (5 × 5 cm).

Digestion with Armillarea mellea protease and purification of peptides on thin-layer plates

10 nmol peptide was digested in 25 #1 0.05 M N-ethyl morpholine acetate pH 8.0 by the Armillarea mellea protease at 35 °C for 24 h. The peptides formed were separated on a cellulose thin-layer plate using electrophoresis in the first dimension at 50 V/cm plate length for 30 min at 15 °C. The electrophoresis buffer used was 10 ~o pyridine, 0.3 ~ acetic acid, pH 6.5. The solvent in the ascending chromatography in the second dimension was n-butanol/acetic acid/water/pyridine (15:3:12:10 by vol.). The peptides were localized by spraying lightly with a solution of 0.1 ~o ninhydrin, 3 ~o 2,4,6-collidine, 10~o acetic acid in ethanol. The cellulose layers with stained peptides were scraped off and the peptides extracted with 50 ~o acetic acid (R. Chert, personal communication).

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Amino acid analysis Peptides were hydrolyzed in constant-boiling hydrochloric acid with 0.1%

phenol at 110 °C for 18-24 h. The tubes were flushed with nitrogen before evacuation and sealed. The amino acid analyses were performed with a Durrum D-500 analyzer.

C-terminal determination Two portions of peptide (1 nmol each) were incubated with carboxypeptidase

Y in 100/~1 50 mM pyridine acetate pH 6.1 at 37 °C. The molar ratio of enzyme to substrate was 1:500. The digestion was stopped at 10 or 60 min by addition of 15 #1 glacial acetic acid and the samples were dried down. The liberated amino acids were identified and quantitated on a Durrum D-500 amino acid analyzer.

Sequencing The amino acid sequences of the peptides were determined by the dansyl

Edman technique [9, 10]. In addition, the Edman degradation was combined with conversion of the isolated thiazolinone derivatives to free amino acids by hydrolysis with hydriodic acid in evacuated, sealed tubes at 130 °C for 20 h [11]. The amino acids were determined on the Durrum analyzer.

Radiolabelling with a251 Radioiodination of porcine glucagon (6 nmol) and gut GLI-1 (2 nmol) was

performed in parallel with the iodide-chloramine-T method, using 100/~Ci of carrier- free solution of 1251- [12]. The iodination was stopped with pyrosulfite and the reaction mixture was placed in an ice bath for 10 min. 20/zl of human albumin 0.1% was added and the proteins precipitated with trichloroacetic acid (final concentration 12.5%).

The mixture was placed in an ice bath for 15 min and centrifuged. The pre- cipitates were washed with 500 #1 0.1 M HC1 in acetone and then with 500/~1 acetone. Residual acetone was carefully blown away with nitrogen.

Autoradiography of peptides on thin-layer plates The 125I-labelled gut GLI-1 and 125I-labelled glucagon together with the added

albumin were degraded by 20 #g a-chymotrypsin in 500/~1 0.5 % NH4HCO3 at 37 °C for 1 h. The digestion was stopped by lyophilization and the digests were redissolved in the electrophoresis buffer: 0.5 % pyridine, 5 % acetic acid, pH 3.5. Aliquots of the digests corresponding to 0.08/~g and 0.8 #g of 125I-labelled glucagon and 12SI-labelled gut GLI-1, respectively, were applied to thin-layer plates. Different peptide maps were made using electrophoresis at pH 6.5 or pH 3.5 in the first dimension and ascending chromatography in the second dimension. The conditions and buffers for electrophoresis and the solvent for chromatography were as described above. The radiolabelled peptides were localized by placing the thin-layer plates in contact with Kodak Blue Brand X-ray film for 19 h.

RESULTS

The gut GLI-I was treated with cyanogen bromide and the peptide mixture was subjected to gel filtration (Fig. 1). Fraction 139-147 contained peptide material

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not absorbing at 280 nm but having Asx as the only N-terminal amino acid residue. Amino acid analysis showed that fraction 143 contained 20 nmol of a pure deca- peptide, the C-terminus of which was determined as -Ile-Ala by treatment of the peptide with carboxypeptidase Y. This C-terminal sequence is the same as the C- terminal sequence of gut GLI-1 and of the proglucagon fragment. As the peptide was devoid of homoserine, which is produced by the cleavage of methionine in peptide chains by cyanogen bromide, the decapeptide must constitute the C-terminus in the intact gut GLI-1.

A b s o r b o n c e ot 280 nrn

0.3

0.2

0.1

0 I [ I 1 ~ 180 200 220 240

I I L I I 1 60 S0 100 120 ~40 160

Froc t ion No.

Fig. 1. Gel filtration of gut GLI-1 treated with cyanogen bromide. Column: SephadexG-50, fine (1.0 × 150 cm); eluent: 5 ~ acetic acid; flow rate: 4 ml/h; fraction size: approx. 500#1.

The amino acid composition and the sequence analysis of the C-terminal decapeptide are summarized in Table I. One portion of the peptide was subjected to Edman degradation, and another portion was digested with the A. mellea protease. Three peptides (designated Am 1 to Am 3) were purified from the digest on thin-layer plates and the amino acid composition and sequences of these peptides confirm the sequence of the intact decapeptide. Only peptide Am 3 contained Asx as the N- terminal amino acid, which places peptide Am 3 at the N-terminus in the decapeptide. The content of lie and Ala in peptide Am 2 places this peptide at the C-terminus. Both Am 1 and Am 2 contained N-terminal Lys, which is consistent with the known specificity of the A. mellea protease which cleaves on the amino side of lysines [13]. The mobility of the peptides Am 1-Am 3 in electrophoresis at pH 6.5 implied that all Asx occurred as Asn [14].

The sequence of the decapeptide shown in Table I differs from the C-terminus of the possible proglucagon fragment of Tager and Steiner [6] only by an inversion between the Lys and Asn at positions 6 and 7 of the decapeptide.

Treatment of the gut GLI-1 and of the C-terminal decapeptide with carboxy- peptidase Y released only alanine and isoleucine and stopped at asparagine. The carboxypeptidase Y is assumed to release all amino acids, including proline, except

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that the release of glycine is slow [15]. This may indicate that the asparagine at the C-terminus of gut GLI-1 is modified in some way, e.g. linked to carbohydrate, and blocks further digestion. However, the carboxypeptidase Y has been found incapable of releasing asparagine placed in the sequence -Lys-His-Asn-lle-Thr-Gln (unpublished results). Therefore it is possible that the sequence -Lys-X-Asn is, in general, a limiting factor in the further release of amino acids by carboxypeptidase Y.

TABLE I

ANALYSIS OF A DECAPEPTIDE FROM THE C-TERMINUS OF PORCINE GUT GLI-1 Fraction no. 143 designates peptide material from the gel filtration experiment with cyanogen- bromide-treated gut GLI-1 (Fig. 1). Am l-Am 3 designate peptides isolated from fraction 143 after treatment with A. mellea protease. The electrophoretic mobility at pH 6.5 was calculated relative to Lys. Amino acid analyses are reported in terms of molar ratios. Notation : ~ Edman degradation/ dansylation; ~ Edman degradation with identification of the thiazolinone derivatives after conver- sion to free amino acid; ~ Residues liberated by carboxypeptidase Y.

Peptide Amino acid composition and sequence Mobility Yield

Fraction no. 143 Asx -Thr -Lys -Arg -Asx -Lys -Asx -Asx -Ile -Ala

o.9~ ~ 2 0.95 0.96 o.9~ 0.95 o.9~ ?.9~ i.o~ 1.o?

Am i Lys -Arg -Asx +0.9 65~

1.00 0.99 1.O6 ) ) )

Am 2 Lys -Asx -Asx -Ile -Ala +0.4 62%

i. O0 1.06 1.o6 1.o0 1.O8 ) ) ) ..... ~ •

Am 3 Asx -Thr O.0 62% 1.o? o.9~

i 5 I0 Final sequence Ash -Thr -Lys -Arg -Ash -Lys -Ash -Asn -Ile -Ala

Fig. 2, a-f, shows autoradiograms of peptide maps of chymotrypsin-treated 12SI-labelled porcine glucagon and l~SI-labelled gut GLI-1. Both Figs. 2a and 2b show two radioactive spots likely to represent the two tyrosine-containing peptides Thr- Ser-Asp-Tyr (7-10) and Ser-Lys-Tyr (11-13) of glucagon which are known to be formed by chymotryptic digestion of glucagon [16]. The electrophoretic mobilities of the peptides at pH 6.5 and pH 3.5 are consistent with the calculated net charges of these chymotryptic peptides.

The autoradiograms of peptide maps of a2SI-labelled gut GLI-1 (Fig. 2c,d) show two dominating spots whose mobility is very similar to the radioactive spots of glucagon. Autoradiograms of peptide maps of a mixture of nSI-labelled glucagon and uSI-labelled gut GLI-1 show the identity of the two major spots of the two pep- tides (Fig. 2e,f), providing evidence that the sequence of residues (7-13) of glucagon is present in gut GLI- I .

t~

O

O E o c-

t J

I

r- {3. o

o

E 2 ¢- U

T Z" O

o

"6 E .o

457

+ 4 - - Elec trophoresis--> + pH 6.5

- 4 -2 0 2 4 I I I I I I = ' ! c m

2 4 6 . .~

+ 4 E l e c t r o p h o r e s i s - - ~ - - pH 3.5

t - ( J

I

fl ~:~ 3

J

c x

-I- ~ Electrophoresis--~ _ pH 6.5

- 4 -2 O-- 2 4 " " r l

+ <- -Electrophoresis- -> ÷ pH 6.5

Fig. 2. Autoradiography of peptide maps o f chymotrypt ic digests o f glucagon (a, b) gut GLI-1 (c,d) and glucagon + gut GLI-1 (e,f), all 12SI-labelled.

458

DISCUSSION

Fig. 3 summarizes the information now available on the amino acid sequence of gut GLI-1 and demonstrates the similarities between pancreatic glucagon, the possible fragment of proglucagon, and gut GLI-1. The dotted bar of gut GLI-1 represents the N-terminal sequence previously determined [5]. The solid bars show the sequence of glucagon also found in the proglucagon fragment. The two chymo- tryptic peptides containing tyrosine and detected both in glucagon and in gut GLI-I are allocated to positions in gut GLI-1 homologous to residues (7-13) in glucagon. This allocation is justified by our knowledge that gut GLI-1 contains an immuno- determinant similar to that of glucagon (2-23). The hatched bars show the C-terminal sequences of the proglucagon fragment and the gut GLI-1 which are analogous. The inversion in this area between Lys and Asn residues is not included in the diagram.

7 11 2729 GLUCAGON

r ~ ' i r

PF:K)GL UCAGON , , , , 37 FRAGMENT ', I , i , , / t , f j ~ ' / ~ . / ~ I

J i I I I I

I :~ _ _ , , , , 1 0 0

GLI-1 I: ' : : ' : ' : ' : ' : ' :1 : : ~ : - : - -RI~ 'T ' fT"~ I

Fig. 3. Schematic outline of the known sequences of glucagon, a possible fragment of proglucagon and gut GLI-1. The sequence of the possible proglucagon fragment as determined by Tager and Steiner [6]. The dotted area of gut GLI-1 represents the N-terminal sequence previously determined [5], and the solid and hatched bars show the sequences presented in this paper.

The C-terminal sequence of the possible proglucagon fragment was determined by Tager & Steiner by treatment of the peptide with carboxypeptidase A and B and determination of the liberated amino acid residues. This method may lead to mis- interpretations in the amino acid sequence [17], and the C-terminal sequences of the gut GLI-1 and the possible proglucagon fragment may be identical. The sequence of the decapeptide from the C-terminus of the gut GLI-1 includes two amino acid residues corresponding to residues 28 and 29 in glucagon. By inference, methionine can be placed in gut GLI-1 as residue eleven counting from the C-terminus, being next to the N-terminal residue in the decapeptide isolated from cyanogen bromide treated gut GLI-1. Residue 27 in glucagon is methionine and thus a three-residue sequence (-Met-Asn-Thr-) in gut GLI-1 is homologous to the C-terminus of glucagon.

The results presented here confirm the relationship between gut GLI-1 and glucagon which has been presented elsewhere [18, 19]. It is proposed that gut GLI-1 contains the full sequence of glucagon and gut GLI-1 does not react with C-terminal specific antiglucagon sera because of structural masking of the C-terminal immuno- determinant. As a consequence of the above structural similarity it is suggested that biosynthesis of gut GLI-1 and glucagon starts with an identical primary gene product. In the pancreatic iz 2 cells this precursor is shortened to glucagon, and in the gut GLI cells of the intestine it is shortened to GLI-1 and perhaps smaller peptides.

ACKNOWLEDGEMENTS

The authors are indebted to D. G. Smyth, National Institute for Medical

459

Research, London, and J. Johansen, Carlsberg Laboratory, Copenhagen, for pro- viding the A. mellea protease and carboxypeptidase Y, respectively, and to E. Geert- sen, Institut for Biokemisk Genetik, University of Copenhagen, for operating the Durrum analyzer. H. Jacobsen and A. Demandt are also grateful to R. Chen, Max- Planck Institut ffir Molekulare Genetik, Berlin, for helpful discussions in his labora- tory of the micromethods of peptide purification and sequencing.

REFERENCES

1 Moody, A. J. (1972) in Glucagon, Molecular Physiology, Clinical and Therapeutic Implications, (Lefebvre, P. J. and Unger, R. H., eds.), 1st edn. pp. 319-341, Pergamon Press, Oxford

2 Heding, L. G., Frandsen, E. K. and Jacobsen, H. (1976) Metabolism 25 Suppl. 1 No. 11, 1327- 1329

3 Polak, J. M., Bloom, S., Coulling, I. and Pearse, A. G. E. (1971) Gut 12, 311-318 4 Unger, R. H., Ohneda, A., Valverde, I., Eisentraut, A. M. and Exton, J. (1968) J. Clin. Invest. 47,

48-65 5 Sundby, F., Jacobsen, H. and Moody, A. J. (1976) Horm. Metab. Res. 8, 366-371 6 Tager, H. S. and Steiner, D. F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2321-2325 7 Johansen, J., Breddam, K. and Ottesen, M. (1975) FEBS 10th Meeting, Paris, abstract No. 740 8 Chen, R., Mende, L. and Arfsten, U. (1975) FEBS. Lett. 59, 96-99 9 Hartley, B. S. (1970) Biochem. J. 119, 805-822

10 Bruton, C. J. and Hartley, B. S. (1970) J. Mol. Biol. 52, 165-178 11 Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M., Gilman, J. G. and Ballantyne, D.

L. (1971) Biochem. 10, 4912--4921 12 Jorgensen, K. H. and Larsen, U. D. (1972) Horm. Metab. Res. 4, 223-224 13 Gregory, H. (1975) FEBS Lett. 51,201-205 14 Offord, R. E. (1966) Nature 211, 591-593 15 Hayashi, R., Moore, S. and Stein, W. H. (1973) J. Biol. Chem. 248, 2296-2302 16 Bromer, W. W., Boucher, M. E. and Koffenberger, Jr., J. E. (1971) J. Biol. Chem. 246, 2822-2827 17 Ambler, R. P. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.) Vol. 11, pp. 155-166,

Academic Press, London 18 Moody, A. J., Frandsen, E. K., Jacobsen, H., Sundby, F. and Orci, L. (1976) Metabolism 25, no.

11, Suppl. 1, 1336-1338 19 Moody, A. J., Jacobsen, H., Sundby, F., Frandsen, E. K. and Orci, L. (1976) in Proc. First Inter-

national Glucagon Symposium, Srinagar, in the press