Molecular Cloning of Neonate/Infant-Specific Pepsinogens from Rat Stomach Mucosa and Their Expressional Change during Development Takashi Kageyama,* ,1 Masao Ichinose,† Shinko Tsukada-Kato,† Masao Omata,† Yuichi Narita,‡ Akihiko Moriyama,§ and Satoshi Yonezawa ¶ *Center for Human Evolutionary Modeling Research, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan; †Department of Gastroenterology, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan; ‡Laboratory of Animal Management, School of Agricultural Sciences, Nagoya University, Nagoya 464-0814, Japan; §Division of Biomolecular Science, Institute of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan; and ¶ Department of Embryology, Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-0392, Japan Received December 6, 1999 To clarify the nature of rat neonate/infant-specific pepsinogens, we carried out their purification and mo- lecular cloning. Prochymosin was found to be the ma- jor neonatal pepsinogen. The general proteolytic ac- tivity of its active form, chymosin, was, however, lower than those of pepsins A and C which are predominant in adult animals. Molecular cloning of rat prochymo- sin cDNA was achieved along with cDNA for another neonate-specific pepsinogen, pepsinogen F, although determination of pepsinogen F in neonatal gastric mu- cosa was unsuccessful, presumably due to its lack of proteolytic activity or different proteolytic specificity. Northern blot analysis confirmed that genes for prochymosin and pepsinogen F are expressed only at neonatal/infant stages and the switching of gene ex- pression to that of pepsinogen C occurred at late in- fant stages. A phylogenetic tree based on nucleotide sequences showed clearly that pepsinogens fall into four major groups, namely prochymosin and pepsino- gen F of the neonate/infant and pepsinogens A and C of adult animals. Although, to date, prochymosin and pepsinogen F were believed to be expressed in only a limited number of mammals, the present results sug- gest that they might be expressed at the neonatal/ infant stage in a variety of mammals. © 2000 Academic Press Pepsinogens are zymogens of pepsins, the major pro- teolytic enzymes in vertebrate gastric juices. They have been purified from the gastric mucosa of various animals and the presence of multiple forms has been demonstrated. To date, pepsinogens have been classi- fied into four major groups, namely, pepsinogen A and pepsinogen C (progastricsin) in adult animals, and prochymosin and pepsinogen F in neonates (1–3). These four groups have been shown to diverge from a common ancestor (4, 5). Pepsinogens A and C are ob- served in many adult animals, except for rodents which express only pepsinogen C, and prochymosin has been demonstrated in some neonatal animals including cow and pig. In primates, such as humans (6) and some monkeys (7), and rodents such as the rat (8), the oc- currence of prochymosin has not been demonstrated, despite extensive investigations. Chymosin, an active form of prochymosin, has been shown to adapt to digest milk proteins (9) and, furthermore, it is thought to be involved with the immune-transfer system from mother to neonate (1). In primates, however, as milk is rich in lactose but poor in proteins, and the bulk of immunity is transferred before birth through the pla- centa (1), chymosin is believed to be less necessary. Indeed, in humans the prochymosin gene has been shown to be inactivated by mutation (6). Rodent milk, in contrast to that of primates, very rich in proteins (10), so the occurrence of prochymosin or neonate- specific digestive proteinases is anticipated. Apart from prochymosin, we demonstrated the occurrence, in rabbit, of the other type of neonatal pepsinogen, namely, pepsinogen F (3). To date, however, it has been unclear whether pepsinogen F is a specific enzyme for neonatal rabbit or it is commonly expressed in stom- achs of other neonatal animals. Developmental changes in the expression of pepsino- gens have been investigated in various animals (11, 12); in rat stomach, mucosal pepsinogen C and its mRNA levels increase dramatically during the period 1 To whom correspondence should be addressed. Fax: +81-568-63- 0085. E-mail: kageyama@pri.kyoto-u.ac.jp. Biochemical and Biophysical Research Communications 267, 806 – 812 (2000) doi:10.1006/bbrc.1999.2047, available online at http://www.idealibrary.com on 806 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.