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Choriolysin H
Choriolysin H (EC 3.4.24.67), also known as high choriolytic enzyme, embryo-specific hatching enzyme, or chorionase, is a metalloendopeptidase enzyme in the astacin family. It is a constituent protease which, together with Choriolysin L, makes up the hatching enzyme and breaks down the embryonic egg envelope, allowing eggs to hatch. It has been studied extensively in medaka (Oryzias latipes), but has been observed in other teleost fishes , including Fundulus heteroclitus. In Oryzias latipes, it has two isoforms that are highly similar and near-indistinguishable in structure and action, but which may be differentiated using High-performance liquid chromatography (HPLC).

Mechanism
Choriolysin H is produced in a larval fish's hatching gland cells within the pharyngeal cavity late in the gastrula stage and assists in hatching by hydrolyzing the egg envelope, or chorion. During egg incubation, the chorion is strengthened by ε(γ-glutamyl)-lysine crosslinks that must be broken for the fish to hatch. While Choriolysin H is unable to break these ε(γ-glutamyl)-lysine bonds directly, it is able to hydrolyze and break other bonds within the envelope, swelling the envelope and exposing sections of the egg envelope that are hydrophobic and may be binding sites for Choriolysin L. This swelling causes the release of multiple polypeptides from the egg envelope, the majority of which are rich in proline, suggesting that breaking bonds between proline and other amino acids is involved in the mechanism of Choriolysin H.

The partial breakdown of the chorion by Choriolysin H facilitates further breakdown by Choriolysin L. The active site contains and is promoted by zinc ions. In Oryzias latipes, the breakdown of the egg envelope is quickest when the ratio of Choriolysin H to Choriolysin L is 20:3, which may have contributed to the large number of copies of the gene that encodes for Choriolysin H.

Choriolysin H is also able to hydrolyze casein and other small molecules using the same active site. It is inhibited by EDTA, and may be reactivated by zinc ions, and to a lesser degree, copper ions. Choriolysin H functions optimally at pH 8 when hydrolyzing the egg envelope and pH 8.7 when hydrolyzing casein.

Structure
Choriolysin H is a single-chain protein. It is synthesized as a preproenzyme about 24 kDa long, and is made up of 270-279 amino acids as a mature enzyme. As a metalloendoprotease, it contains calcium, copper, magnesium and zinc. In Oryzias latipes and Fundulus heteroclitus, there are multiple copies of the gene for Choriolysin H, all of which lack introns. The lack of introns could be due to retroposon duplication, but this is unlikely as retroposons generally lack promoters, and the genes in Oryzias latipes and Fundulus heteroclitus have promotors for each copy of the Choriolysin H gene. The gene that codes for Choriolysin L does have introns, so the loss of introns likely occurred after the evolutionary divergence of Choriolysin H and Choriolysin L.

Isomers in Oryzias latipes
In Oryzias latipedes, there are two isoforms of Choriolysin H. These isomers can be distinguished from one another with HPLC, but are indistinguishable with other methods due to their similarity. Between the two isomers, 96% of the amino acid sequence is shared. The two isoforms of Choriolysin H have the same ability to hydrolyze the inner membrane of the egg envelope, as well as having near-identical affinities for substrates, molecular weights, amino acid makeups, and, when incubated together, the ratio of the two isomers to each other stays constant, suggesting that they are highly similar to the point that they are considered a single enzyme.

In Oryzias latipes, there are eight copies of the gene for producing Choriolysin H, which were likely produced by an unequal crossing-over recombination event and subsequent inbreeding, and may have contributed to the two slightly differing isoforms of the enzyme. Of the eight copies of the gene, five code for one isomer (HCE23) and three code for the other (HCE21), and these are distributed in the genome unevenly. Three repeats of each gene are clustered within a 17kb section, and another two copies of gene HCE23 are located over 15kb away. The high similarity of all eight gene copies means that the same primers can be used for all, as the transcription sites for each are highly conserved and each has a promoter segment that includes a TATA box. The gene for HCE21 is nine amino acids longer than that of HCE23, due to a slightly longer propeptide region.

Presence in other organisms
Choriolysin H is most well-characterized in Oryzias latipes, but it is also present in other teleosts. Not all teleosts have both Choriolysin H and Choriolysin L, but many have one of the two enzymes or a species-specific homolog. Fundulus heteroclitus, the mummichog, has both Choriolysin H and Choriolysin L, and Choriolysin H has multiple gene copies, each with no introns. Choriolysin H is highly conserved between Oryzias latipes and Fundulus heteroclitus, and Choriolysin H is able to swell the egg envelope in Fundulus heteroclitus eggs.

Choriolysin H is also present in Oncorhynchus masou, Danio rerio, and in Chrysiptera parasema. Though these organisms have no homolog for Choriolysin L, the genetic code for Choriolysin H is phylogenetically similar to Oryzias latipes, and so these four organisms are considered to be a single clade. The similarity between the genetic code for Choriolysin H of these organisms despite them not being closely related suggests that Choriolysin H is highly conserved among teleosts.

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