Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes

Abstract

BackgroundDuplication 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.ResultsThe 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.ConclusionsWe 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.

Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis

“…As a result of hardening, the fertilized egg envelope becomes insoluble in denaturing reagents, such as urea and SDS. In contrast, the unfertilized egg envelope is generally solubilized by boiling in those reagents, and the component proteins (ZP proteins) dissociate into their monomeric forms (Sano et al., , , ; Yasumasu et al., ). However, the unfertilized Pacific herring egg envelopes did not dissolve completely by boiling in SDS, suggesting that they were partially hardened in the developing oocyte before fertilization.…”

An evolutionary insight into the hatching strategies of pipefish and seahorse embryos

“…According to Sano et al (), ancestral teleostean hatching enzyme is considered to have only a swelling effect. Thus, ancestral teleostean embryos could hatch out by swelling and softening the egg envelope and rupturing it by the subsequent movement of embryos.…”

Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos

“…The zebrafish genome encodes 2 HEs (zHE1 and zHE2). zHE1 mRNA is detected as early as 19 hpf and is no longer detected post-hatch, but zHE2 is not expressed at these time points [22,23]. There is evidence of sub-functionalization of HE paralogues in some teleost lineages, but zHE2 is not thought to be relevant for hatching in zebrafish [23]; we will not consider it further.…”

“…zHE1 mRNA is detected as early as 19 hpf and is no longer detected post-hatch, but zHE2 is not expressed at these time points [22,23]. There is evidence of sub-functionalization of HE paralogues in some teleost lineages, but zHE2 is not thought to be relevant for hatching in zebrafish [23]; we will not consider it further. Purified recombinant activated zHE1 degrades ZP proteins from chorions of unfertilized eggs and fertilized embryos at specific cleavage sites in vitro [22], but it remains unclear how the activity of this enzyme is regulated in vivo.…”

“…HE is an astacin-like metalloprotease with a zinc-dependent catalytic domain produced by cells of the hatching gland [22][23][24]. As with other metalloproteases, HE has an auto-inhibitory N-terminal propeptide that must be proteolytically cleaved to activate the zymogen [25].…”