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Purification and Sequence of Rat Oxyntomodulin (Enteroglucagon/Peptide/Intestine/Proglucagon/Radlolmmunoassay) NATHAN L

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Purification and Sequence of Rat Oxyntomodulin (Enteroglucagon/Peptide/Intestine/Proglucagon/Radlolmmunoassay) NATHAN L Proc. Nati. Acad. Sci. USA Vol. 91, pp. 9362-9366, September 1994 Biochemistry Purification and sequence of rat oxyntomodulin (enteroglucagon/peptide/intestine/proglucagon/radlolmmunoassay) NATHAN L. COLLIE*t, JOHN H. WALSHO, HELEN C. WONG*, JOHN E. SHIVELY§, MIKE T. DAVIS§, TERRY D. LEE§, AND JOSEPH R. REEVE, JR.t *Department of Physiology, School of Medicine, University of California, Los Angeles, CA 90024; *Center for Ulcer Research and Education, Gastroenteric Biology Center, Department of Medicine, Veterans Administration Wadsworth Center, School of Medicine, University of California, Los Angeles, CA 90073; and §Division of Immunology, Beckman Institute of City of Hope Research Institute, Duarte, CA 91010 Communicated by Jared M. Diamond, May 26, 1994 ABSTRACT Structural information about rat enteroglu- glucagon plus two glucagon-like sequences (GLP-1 and -2) cagon, intestinal peptides containing the pancreatic glucagon arranged in tandem. The present study concerns the enter- sequence, has been based previously on cDNA, immunologic, oglucagon portion of proglucagon (i.e., the N-terminal 69 and chromatographic data. Our interests in testing the phys- residues and its potential cleavage fragments). iological actions of synthetic enteroglucagon peptides in rats Our use of the term "enteroglucagon" refers to intestinal required that we identify precisely the forms present in vivo. peptides containing the pancreatic glucagon sequence. Fig. 1 From knowledge of the proglucagon gene sequence, we syn- shows two proposed enteroglucagon forms, proglucagon-(1- thesized an enteroglucagon C-terminal octapeptide common to 69) (glicentin) and proglucagon-(33-69) (OXN; see Fig. 1). both proposed enteroglucagon forms, glicentin and oxynto- The primary structures based on amino acid sequence data of modulin, but sharing no sequence overlap with glucagon. We pig glicentin (6), ofdog glicentin and OXN (7), and ofalligator then developed a radilmmunoassay using antibodies raised gar (the holostean fish Lepisosteus spatula) OXN (8) have against the octapeptide that was specific for enteroglucagon been published. By contrast, information in the rat and peptides without cross-reacting with glucagon. Rat intestine human has relied upon a combination of cDNA, immuno- was extracted, and one presumptive enteroglucagon form was logic, and chromatographic techniques (9-11). However, the purified by following the enteroglucagon C-terminal octapep- peptide structure of either enteroglucagon form has not been tide-like immunoreactivity through several HPLC purification fully characterized in the rat and human. steps. Structural characterization ofthe material by amino acid We used a strategy similar to that of Blache et al. (12) to composition, microsequence, and mass spectral analyses iden- generate antibodies to the C-terminal octapeptide ECO com- tified the peptide as rat oxyntomodulin. The 37-residue peptide mon to both enteroglucagon forms, whose sequence was consists of pancreatic glucagon plus the C-terminal extension, deduced from the proglucagon cDNA sequence. This anti- Lys-Arg-Asn-Arg-Asn-Asn-Ule-Ala. This now permits synthesis body would then recognize both presumptive enteroglucagon ofan unambiguous duplicate ofendogenous rat oxyntomodulin forms without cross-reacting with glucagon. Our goal in this for physiological studies. study was to develop such a sequence-specific RIA, to isolate rat enteroglucagon peptides, and then to characterize their Gene cloning techniques have accelerated regulatory peptide peptide structure. research by providing rapid sequence information about potential physiological signals. In particular, we are inter- MATERIALS AND METHODS ested in those signals that regulate intestinal growth and nutrient absorption. Strong but circumstantial evidence has Synthetic Peptides. Two peptides for this study were syn- implicated the hormone enteroglucagon, secreted by the thesized in the Peptide Biochemistry Core Facility of the intestine, as a signal for intestinal adaptation (1-3). Despite Veterans Administration/University of California at Los the cloning of the gene for enteroglucagon's precursor, Angeles Gastroenteric Biology Center: Lys-Arg-Asn-Arg- proglucagon (4), the endogenous forms ofrat enteroglucagon Asn-Asn-Ile-Ala (rat ECO) and Tyr-Lys-Arg-Asn-Arg-Asn- have not been fully characterized. We emphasize that only Asn-Ile-Ala (D-Tyr-ECO). These peptides were made on a the potential structures of the active peptides are derived Biosearch Sam II peptide synthesizer using tert-butoxycar- from the gene sequence because of the numerous processing bonyl-amino acid coupling strategies, cleaved with hydrogen steps that intervene between gene transcription and peptide fluoride, and purified by reverse-phase HPLC. Fractions of end product. Within a single species, tissue-specific differ- >90%o purity were pooled, lyophilized, and shown to have the ences in proglucagon processing, such as that occurring in the correct composition by amino acid and mass spectral anal- pancreas and intestine, further add to the structural hetero- ysis. Synthetic peptides for RIA cross-reactivity tests [hu- geneity of secreted peptides. These considerations place a man/rat/porcine vasoactive intestinal peptide (VIP), human premium on full structural characterization of peptides if we gastric inhibitory peptide (GIP), porcine OXN, glucagon, are to determine their precise physiological roles. Hence, our human gastrin, human GLP-1-(7-36) fragment, and secretin] study of enteroglucagon function in rats began with the were from Peninsula Laboratories. objective of unambiguously identifying the molecular forms HPLC Columns. Preparative HPLC C18 and C8 columns of this gut hormone. (21.4 mm x 25 cm; C18 83-223-C and C8 83-323-C) were Fig. 1 summarizes the structure and nomenclature of the obtained from Rainin (Woburn, MA). The semipreparative proglucagon precursor predicted from the cloned rat gene Vydac C4 (10 mm x 25 cm; 214 TP510) and the analytical sequence (4, 5). From the nucleic acid sequence, rat proglu- cagon is a 160-residue polypeptide containing pancreatic Abbreviations: ECO, enteroglucagon C-terminal octapeptide; ECO- LI, ECO-like immunoreactive material; OXN, oxyntomodulin; GLP-1 and -2, glucagon-like peptides 1 and 2. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at the present payment. This article must therefore be hereby marked "advertisement" address: Department of Biological Sciences and Institute for Bio- in accordance with 18 U.S.C. §1734 solely to indicate this fact. technology, Texas Tech University, Lubbock, TX 79409. 9362 Downloaded by guest on October 2, 2021 Biochemistry: Collie et al. Proc. Nati. Acad. Sci. USA 91 (1994) 9363 PROGLU'CAGON were collected for columns of 2.1-cm diameter and larger. Fractions of4 ml were collected for 1.0-cm columns, and 1-ml NH (;ICr,(ENTI]N OXN fractions were collected for all other columns. I1- H Peptide Structurl Analyses. For amino acid analysis, a GRPP small portion (50-200 pmol) of the purified ECO-like immu- 30 noreactive material, which we call ECO-LI, was dried down F in borosilicate tubes (6 x 30 mm, previously heated to 4000C 33 T G I S for 24 hr). The tubes were placed into a hydrolysis chamber L D with 300 /4 of HCl containing 0.2% 2-mercaptoethanol and U heated to 1100C for 24 hr. After cooling, the HCl vapors were C K A removed by vacuum, and the amino acids were dissolved in G L Beckman amino acid dilution buffer and analyzed on a 0 Beckman 6300 amino acid analyzer. Data were collected on N R 61_ R a Nelson analytical data system. 64-- Purified ECO-LI was sequenced on a City of Hope gas- IP-1 69-, F phase microsequencer as described (13). Phenylthiohydan- 72'- FIc( toin derivatives ofamino acids were analyzed as described by Hawke et al. (14). GLP-1 L For mass spectral analysis, the purified peptide (25-100 NA K pmol) was analyzed with a JEOL HX 100HF soluble focusing 1081 T R magnetic sector mass spectrometer operating at 5-kV accel- 110- IP-2 erating potential. Sample ionization was accomplished by 123,. N a 6000-eV xenon atom beam. in R using Dry samples 1.5-ml 126- N polypropylene Microfuge tubes were taken up in 1-2/4 of5% N aqueous acetic acid and added to -1 /4 of the liquid matrix GLP-2 on a 1.5 x 6.0 mm stainless steel stage at the tip of a direct A insertion probe. The liquid matrix consisted ofdithiothreitol/ 160 dithioerythritol, 5:1 (vol/vol at room temperature), spiked with 6 mM camphorsulfonic acid. Positive-ion spectra were 01l collected under computer control using a JEOL DA5000 data system. Multiple scans over the mass range m/z = 100-3500 FIG. 1. Rat proglucagon (left-most vertical bar) undergoes ex- (cycle time, 45 s) were collected. tensive posttranslational processing in the intestine, yielding four Intetnal Extraction. Distal small intestine (jejunoileum) peptides related in sequence to pancreatic glucagon: glicentin and was at sacrifice from male rats oxyntomodulin (OXN), both containing the entire glucagon se- obtained Sprague-Dawley quence, as well as glucagon-like peptides 1 and 2 (GLP-1, GLP-2), (250- to 350-g body weight), rinsed with ice-cold Kreb's the latter two sharing 50-70%o homology to glucagon. Putative sites bicarbonate Ringer solution (pH 7.4), cut open longitudinally, of proteolytic cleavage are indicated by horizontal parallel lines blotted gently, snap-frozen on dry ice, and stored at -70°C (numbered residues) that border pairs of basic amino acids. The until extraction. Frozen intestinal tissue (107 g) was homog- vertical bars labeled OXN and ECO show the presumptive sequence enized in 4% aqueous CF3COOH (1 liter) at 4°C with a (given in single-letter amino acid code) for OXN deduced from the rat Polytron homogenizer. The extract was then centrifuged at proglucagon gene sequence (see ref. 4). Rat OXN is thus predicted 10,000 x g for 30 min, and the supernatant was applied to a to consist of glucagon plus enteroglucagon C-terminal octapeptide HPLC column. (ECO). GRPP, glicentin-related pancreatic peptide; IP-1 and -2, preparative intervening peptides 1 and 2.
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