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J Mol Evol (1992) 34:536-543 Journal of Molecular Evolution @ Springer-Verlag New York Inc. 1992 Evolutionary Origin of a Kunitz-Type Trypsin Inhibitor Domain Inserted in the Amyloid/3 Precursor Protein of Alzheimer's Disease Kazuho Ikeo, ~,3 Kei Takahashi, 2 and Takashi GojoborP i DNA Research Center, National Institute of Genetics, Mishima 411, Japan 2 Department of Physiology, Shimane Medical University, Izurno 693, Japan 3 The Graduate University for Advanced Studies, Mishima 411, Japan Summary. The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid/3 precursor protein (/3APP). It is known that/3APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhib- itors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present/3APP gene di- verged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in /3APP have an evolutionarily close relationship with the inter-a-trypsin inhibitor or trypstatin, which in- hibits the activity of tryptase, a novel membrane- bound serine protease in human T4 + lymphocytes. Key words: Serine protease inhibitor -- Kunitz type -- Evolutionary origin -- Alzheimer's disease -- Insertion sequence Offprint requests to: T. Gojobori Introduction The Kunitz-type inhibitor is one of the serine pro- tease inhibitors. It usually has a low relative mo- lecular mass, a basic isoelectric point, and one or several inhibitory domains (Salvessen and Nagase 1989). This inhibitor was first isolated as a trypsin inhibitor from bovine pancreas (Laskowski et al. 1974). It has an amino acid sequence of 58 residues with three disulfide bridges. The pattern of its di- sulfide bridges is the same as that in the kringle domains of serine proteases, which are involved with the blood coagulation cascade (Ikeo et al. 1991). The enzymatic reaction between a protease and its inhibitor is characterized by the formation of a pseudoirreversible inhibitor-protease complex (Laskowski and Kato 1980). Although the Kunitz- type inhibitor is found in virtually all tissues and has a wide range of specificity (Fioretti et al. 1983), very little is known about the biosynthesis and phys- iological function of the Kunitz-type inhibitor. This inhibitor has also been found in snake venom and in some insects. In the last few years, it has become apparent that various proteins contain an insertion of the Kunitz- type inhibitor domain. It is very likely that these proteins were assembled as a consequence of exon shuffling through incorporation of genetic elements or domains from different evolutionary precursors. The Kunitz-type inhibitor sequence was found in a precursor protein of the amyloid/3 protein, which is a component of cerebral depositions in the brains of patients with Alzheimer's disease (Fig. 1). Alzheimer's disease is the most common neu- rodegenerative disorder, affecting over 2 million