title: Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes.
abstract: BACKGROUND
  Duplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process.
  
  
  RESULTS
  The milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region.
  
  
  CONCLUSIONS
  We infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.
authors.affiliation: Department of Chemistry, Faculty of Science, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295, Japan. kaori-s@josai.ac.jp.
authors.forename: Kaori
authors.initials: K
authors.lastname: Sano
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo, 102-8554, Japan. k-mari@sophia.ac.jp.
authors.forename: Mari
authors.initials: M
authors.lastname: Kawaguchi
authors.affiliation: National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie, 516-0193, Japan. swat@affrc.go.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo, 102-8554, Japan. s-yasuma@sophia.ac.jp.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Yasumasu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D004527:Egg Proteins / Q000378:metabolism; D019143:Evolution, Molecular; D005399:Fishes / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D020131:Genes, Duplicate; D006023:Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D004527:Egg Proteins; D006023:Glycoproteins; D008666:Metalloendopeptidases; C024344:envelysin