J. Biochem. 103, 290-296 (1988) Molecular Cloning and the Nucleotide Sequence of cDNA for Embryonic Chicken Pepsinogen: Phylogenetic Relationship with Prochymosin1 Kensuke Hayashi, * Kiyokazu Agata, * * Makoto Mochii, * * Sadao Yasugi, * Goro Eguehi, and Takeo Mizuno* *Zoological Institute , Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113; and **Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Aichi 444 Received for publication, September 17, 1987 Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a eDNA clone by screening a Agtll library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this eDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequenc ing longer cDNAs obtained by screening the same library with the 200-base pair eDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other. Pepsinogen is an inactive zymogen of pepsin, which is found in rat (16). Although the physiological functions of almost universally in the stomachs of vertebrates (1, 2). chymosin are not yet clear, chymosins are predominant in Gastric aspartyl proteinases are classified into three neonatal mammals and very minor in adult. groups, that is, pepsinogen A, prochymosin, and progastric Previously, embryonic chicken pepsinogen (ECPg) was sin (pepsinogen C), with respect to their enzymatic and purified and characterized by Yasugi and Mizuno (17). It is immunochemical features. The complete amino acid secreted only during the embryonic period. In chicken sequences of the following proteins have been determined: proventriculus ECPg synthesis begins on day 10 of incuba pepsinogen A of human (3), Japanese monkey (4), pig (5, tion and ceases by day 20 (18). It is a unique pepsinogen 6), and adult chicken (7), progastricsin of Japanese molecule with the large molecular weight of 56,000 (17), monkey (8) and rat (9), and bovine prochymosin (10). The whereas the molecular weights of other pepsinogens ex molecular phylogenetic tree constructed for these se amined so far are less than 48,000. quences revealed that progastricsin diverged first from To investigate the phylogenetic relationship between their common ancestral pepsinogen, and then prochymosin ECPg and prochymosin, we isolated cDNA clones for ECPg and pepsinogen A diverged (8, 11). These studies suggest and determined the primary structure of ECPg. These that the chicken diverged from mammals immediately cDNA clones will be useful for the investigation of the after prochymosin diverged from pepsinogen A. If so, the mechanisms of tissue and stage-specific expression of common ancester of chicken and cow had to possess an ECPg in the embryonic proventricular epithelium, which is ancestral chymosin-type proteinase, and the modern chi known to be induced by proventricular mesenchyme (19, cken is expected to possess a chymosin type-proteinase. 20). Chymosin, which has the characteristic of high milk clotting activity, for a long time could be demonstrated only MATERIALS AND METHODS in neonatal calf (12) In this decade, chymosin-like proteinases were reported for other neonatal animals such Antiserum and Western Blot Analysis-Antiserum as pig (13), cat (14), and seal (15). Higher milk clotting (anti-ECPg) for screening the library was raised in a activity in neonatal gastric juice than in adult was reported BALB/c mouse immunized with ECPg, which was purified from 15-day chicken proventriculi according to Yasugi and 1 This work was carried out under the NIBB Cooperative Research Mizuno (17). The monoclonal antibody against ECPg (Y37) Program (85-134). It was also supported by a Grant-in-Aid (No. 61540513) from the Ministry of Education, Science and Culture of was prepared as described (21). Western blotting was Japan. performed as described by Towbin et al. (22). Briefly, Abbreviations: ECPg, embryonic chicken pepsinogen; SDS, sodium about 10 ƒÊg of protein was subjected to SDS-polyacryl dodecyl sulfate; SSC, 0.15 M NaCI-0.015 M sodium citrate. amide gel electrophoresis (23) and subsequently electro- 290 J. Biochem. Nucleotide Sequence of Embryonic Chicken Pepsinogen cDNA 291 phoretically transferred onto a nitrocellulose filter (Bio Construction of Phylogenetic Tree-The mutation dis Rad). Antigens were detected with the mouse anti-ECPg tances among the amino acid sequences of gastric aspartyl antibody and horseradish peroxidase-labeled rabbit anti proteinases were calculated according to Fitch and Mar body against mouse IgG. goliash (30), except that codon ATA was regarded to code Construction and Screening of ƒÉgt11 cDNA Library for isoleucine. The methods of Fitch and Yasunobu (31) -The methods for the construction and screening of the were used for aligning the sequences with gaps and for library were those previously described (24). The poly(A) I weighting the gaps. The phylogenetic trees were construct RNA was prepared by oligo(dT) -cellulose chromatography ed from the matrix of mutation distances by the Fitch and from the total RNA of 12-day embryonic chicken pro Margoliash method (30), with a modification by Fitch and ventriculi. The cDNA was synthesized from this poly(A)+ Yasunobu (31), in which each side of a bifurcation receives RNA with oligo(dT) as primer. The double-stranded cDNA equal weight. was ligated with EcoRI linkers, and was inserted into the EcoRI site of ƒÉgtll. The library was amplified and RESULTSAND DISCUSSION screened according to Young and Davis (25). The cDNA clone (named as ECP200) selected with anti-ECPg antibody Characterization of the Anti-ECPg Antibody-The cross was used to rescreen the same library. Plaque hybridiza reactivity of antiserum raised against purified ECPg (anti tion was carried out according to Maniatis et al . (26). ECPg) which was used for screening of the cDNA library is Northern Blot Analysis-Poly(A)+ RNA purified by shown in Fig. 1. Crude extracts from four organs of oligo(dT)-cellulose column chromatography was denatured in glyoxal, electrophoresed in 1% agarose gels with poly(A) RNA as a size marker, and blotted on a nylon membrane (Pall) in 20•~SSC (1•~SSC, 0.15 M NaCl-0.015 M sodium citrate). Radio labeling of the probes was performed by nick translation with a-[32P]dCTP (double stranded DNA) or by 5•Œ-labeling with ƒÁ-[32P]ATP and polynucleotide kinase (single-stranded DNA). Blots were hybridized, washed and exposed for autoradiography ac cording to Maniatis et al. (26). DNA Sequencing and Primer Extension-The entire cDNA inserts and their appropriate restriction fragments were subcloned into pUC18. Sequencing was performed by the dideoxy chain termination method (27) after denatura tion of double-stranded plasmid DNA with NaOH. About 90% nucleotides were determined from both strands. Primer extension was carried out as described by Nathans and Hogness (28). A 20-mer synthetic primer was labeled with [32P]ATP and RNase-free T4 kinase (Takara Shuzo). The mixture of 20ƒÊg of 15-day proventricular Fig. 1. Western blot analysis displaying the cross reactivity poly(A)+ RNA and 32P-labeled primer was heated at 90•Ž of anti-ECPg. Ten micrograms protein of each crude extract from for 3 min. Then annealing was performed at 30•Ž for 30 esophagus (E), proventriculus (P), gizzard (G), small intestine (S) of min after 1 pl of 3 M KCl was added to the mixture. cDNA 15-day chicken embryos was loaded on SDS-polyacrylamide gel electrophoresis and blotted on nitrocellulose membrane. Pep was synthesized with reverse transcriptase (Promega sinogens were immunochemically detected with anti-ECPg antiserum Biotec) at 30•Ž for 10 min, and then at 40•Ž for 80 min. (the four left lanes) or with monoclonal antibody Y37 (far right lane). The extended products were eluted from the sequencing gel Molecular weight markers were albumin (66,000) and ovalbumin and sequenced by the Maxam-Gilbert method (29). (45,000). Fig. 2. The deduced amino acid sequences of ECPg and other gastric aspartyl proteinases. The residues are numbered from the amino-terminus of embryonic chicken prepepsinogen. The boxed residues are identical with ECPg. Panels A and B: The sequences around the two active-site aspartic acids (asterisks). Panel C: The sequences of the 18 amino acids at the carboxyl termini. The sequence data were obtained from the following references: pepsinogen A of human (Human A) (3), of monkey (Monkey A) (4), of pig (Porcine A) (5, 6), of adult chicken (Chicken A) (7), bovine prochymosin (Bovine C) (10), progastricsin of monkey (Monkey G) (8), and of rat (Rat G) (9). Vol. 103, No. 2, 1988 292 K. Hayashi et al. Fig. 3. Northern blot analysis. Poly(A)+RNA was extracted from 15-day proventriculus (E) and adult proventriculus (A). One microgram of each RNA was denatured in glyoxal, subjected to 1% agarose gel electrophoresis, and blotted on nylon membrane in 20x SSC. Hybridization was performed at 50•Ž overnight after prehy bridization with denatured salmon sperm DNA for more than 4 h. The blot was washed twice in 2•~SSC, 0.1% SDS (sodium dodecyl sulfate) at 37•Ž for 30 min and twice in 1•~SSC, 0.1% SDS at room temperature for 30 min.