he1.2 (hatching enzyme 1.2, ZHE1/zHCE-1) encodes the zebrafish hatching enzyme, a secreted astacin-family (M12A) zinc metalloendopeptidase (EC 3.4.24.67, choriolysin H-like). It is synthesized as a preproenzyme with a signal peptide and an activation propeptide by the hatching gland cells, which originate in the polster and come to lie in the epidermis of the anterior yolk sac; expression peaks in pre-hatching embryos and disappears after hatching. The mature enzyme is released into the perivitelline/hatching liquid, where it cleaves the N-terminal regions of the egg-envelope (chorion) zona pellucida glycoproteins ZP2 and ZP3 at defined sites (major site TVQQS-|-DYLIK, minor site KLMLK-|-APEPF), swelling and partially digesting the envelope so that the embryo can emerge. Unlike medaka, which uses a cooperating HCE/LCE pair, zebrafish hatching is performed by this single enzyme; the paralog ZHE2 is barely expressed. A 1.10 A crystal structure shows a monomeric, mitten-shaped protein whose central cleft carries the catalytic zinc and determines substrate recognition (Arg182 salt-bridging the P1' Asp of the major substrate).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004222 metalloendopeptidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT propagation of metalloendopeptidase activity from the astacin/M12A ancestral node (PTN000013301). For he1.2 itself the activity is established directly: purified ZHE1 cleaves egg-envelope ZP2/ZP3 at six internal N-terminal sites (PMID:19021768) and the crystal structure shows a zinc metalloprotease active-site cleft (PMID:20727360). UniProt assigns EC 3.4.24.67. Reason: he1.2 sits inside the astacin clade that inherited the M12A metalloendopeptidase fold, and the target has its own experimental grounding (the ZFIN entry appears in the WITH/FROM list as expected). The cleavage sites mapped in PMID:19021768 lie within the ZP2/ZP3 polypeptides, i.e. this is endopeptidase rather than exopeptidase activity, and the enzyme requires a catalytic Zn(2+) (UniProt COFACTOR). This is the core activity of the gene product. Supporting Evidence: PMID:20727360 Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. PMID:19021768 The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. file:DANRE/he1.2/he1.2-uniprot.txt FUNCTION: Metalloendopeptidase which participates in the breakdown of the egg envelope at the time of hatching. Cleaves the N-terminal regions of the zona pellucia glycoproteins ZP2 and ZP3 |
| GO:0004222 metalloendopeptidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Peptidase M12A (astacin) domain signature IPR001506, which spans residues 65-263 of the mature enzyme. The same activity is demonstrated experimentally for he1.2 (PMID:19021768, PMID:20727360). Reason: The astacin-domain mapping is correct for this protein: the domain contains the HExxHxxGFxHExxRxDR zinc-binding motif (His163 is the catalytic zinc ligand in UniProt FT BINDING) and the enzyme's endopeptidase activity on ZP2/ZP3 is proven. Redundant with the IDA row but not wrong. Supporting Evidence: file:DANRE/he1.2/he1.2-uniprot.txt CATALYTIC ACTIVITY: Reaction=Hydrolysis of the inner layer of fish egg envelope. Also hydrolysis of casein and small molecule substrates such as succinyl-Leu-Leu-Val-Tyr-|-7-(4-methyl)coumarylamide.; EC=3.4.24.67 PMID:20727360 The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SUBCELLULAR LOCATION "Secreted" (SL-0243) mapped to extracellular region. The location is experimentally grounded for he1.2: ZHE1 was purified from the hatching liquid of pre-hatching embryos (PMID:19021768), and the precursor carries a signal peptide (residues 1-19) and activation propeptide. Reason: The hatching enzyme must act outside the embryo, on the chorion that encloses it; purification of the active enzyme from hatching liquid is direct evidence of secretion. extracellular region is the correct GO term for this compartment. Supporting Evidence: file:DANRE/he1.2/he1.2-uniprot.txt SUBCELLULAR LOCATION: Secreted PMID:19021768 one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping of the astacin (IPR001506) and metallopeptidase (IPR006026) signatures to proteolysis. he1.2 demonstrably hydrolyses egg-envelope proteins ZP2 and ZP3 (PMID:19021768). Reason: Proteolysis is exactly the process this enzyme carries out: it cleaves peptide bonds in the chorion subunit proteins, partially digesting and swelling the envelope. The term is broad but it is the direct process of the catalytic activity, not a downstream phenotype. Supporting Evidence: PMID:19021768 The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. file:DANRE/he1.2/he1.2-uniprot.txt FUNCTION: Metalloendopeptidase which participates in the breakdown of the egg envelope at the time of hatching. |
| GO:0008237 metallopeptidase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the generic metallopeptidase signatures IPR006026/IPR024079. True but less specific than what is known: he1.2 is an endopeptidase that cleaves internal sites in ZP2/ZP3 (PMID:19021768). Reason: metallopeptidase activity is the parent of metalloendopeptidase activity. Because the cleavage sites are internal to the ZP2/ZP3 polypeptides and the enzyme is an astacin (EC 3.4.24.67), the child term GO:0004222, which the gene already carries by IDA and IBA, is the informative annotation; the parent adds nothing. Proposed replacements: metalloendopeptidase activity Supporting Evidence: PMID:19021768 The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. file:DANRE/he1.2/he1.2-uniprot.txt FUNCTION: Metalloendopeptidase which participates in the breakdown of the egg envelope at the time of hatching. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the metallopeptidase zinc-binding signature IPR006026. he1.2 binds one catalytic Zn(2+) per subunit via His163 (UniProt COFACTOR/BINDING), and the crystal structure is of a zinc metalloprotease (PMID:20727360). Reason: Zinc binding is real but is the cofactor requirement of the catalytic activity rather than a function in its own right; the informative annotation is metalloendopeptidase activity, of which the zinc site is part. Keep as a supporting, non-core annotation. Supporting Evidence: file:DANRE/he1.2/he1.2-uniprot.txt COFACTOR: Name=Zn(2+); Xref=ChEBI:CHEBI:29105 PMID:20727360 Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. file:DANRE/he1.2/he1.2-deep-research-falcon.md its activity can be inhibited by chelation of divalent metals (EDTA), consistent with dependence on a catalytic Zn |
| GO:0005576 extracellular region | EXP PMID:19021768 Purification and characterization of zebrafish hatching enzy... | ACCEPT | Summary: UniProt EXP annotation from Sano et al. 2008, who purified ZHE1 from the hatching liquid of zebrafish embryos and confirmed by protein-level analysis that it is the single secreted hatching enzyme. The cached record is abstract-only. Reason: Purification of the enzyme from hatching liquid is direct evidence that the mature protein is extracellular; UniProt also uses this paper as the evidence for the "Secreted" subcellular location and for the mature chain boundary (residue 65 onward, by direct protein sequencing). The abstract states the key fact explicitly, so no deference beyond the abstract is needed. Supporting Evidence: PMID:19021768 one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid file:DANRE/he1.2/he1.2-uniprot.txt SUBCELLULAR LOCATION: Secreted |
| GO:0004222 metalloendopeptidase activity | IDA PMID:20727360 Crystal structure of zebrafish hatching enzyme 1 from the ze... | ACCEPT | Summary: UniProt IDA from the 1.10 A crystal structure of ZHE1 (Okada et al. 2010). The structure defines the active-site cleft, and alanine-scanning of the two substrate peptides maps the residues (P4-P2' of the major site, GluP3' of the minor site) recognized by the enzyme, with Arg182 forming a salt bridge to the P1' Asp. Abstract-only cache. Reason: The paper directly characterizes the catalytic machinery and substrate recognition of the zebrafish enzyme, and the substrates are internal peptides derived from the ZP2/ZP3 cleavage sites, so the activity is metalloendopeptidase activity. This is the core molecular function; consistent with the IBA and IEA rows. Supporting Evidence: PMID:20727360 Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. PMID:20727360 Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1' contributes the most and that the residues at P4-P2' also contribute to the recognition of the major substrate peptide by ZHE1 |
| GO:0006508 proteolysis | IDA PMID:20727360 Crystal structure of zebrafish hatching enzyme 1 from the ze... | ACCEPT | Summary: UniProt IDA for proteolysis from the ZHE1 crystal-structure paper, which combines the structure with substrate-peptide mutagenesis to explain how the enzyme recognizes and cleaves its two egg-envelope substrate peptides. Reason: The enzyme itself performs the peptide-bond hydrolysis assayed in this paper, so proteolysis is the direct process of its activity. Consistent with the PMID:19021768 and IEA rows for the same term. Supporting Evidence: PMID:20727360 The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. file:DANRE/he1.2/he1.2-uniprot.txt CATALYTIC ACTIVITY: Reaction=Hydrolysis of the inner layer of fish egg envelope. |
| GO:0008270 zinc ion binding | IDA PMID:20727360 Crystal structure of zebrafish hatching enzyme 1 from the ze... | KEEP AS NON CORE | Summary: UniProt IDA for zinc binding from the crystal structure, which resolves the catalytic zinc in the central cleft of this zinc metalloprotease. UniProt records one zinc per subunit, ligated by His163 (FT BINDING). Reason: The zinc site is experimentally observed, so the annotation is correct, but it describes the cofactor of the catalytic activity rather than a separate function of he1.2. Kept as non-core, matching the IEA row for the same term. Supporting Evidence: PMID:20727360 Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. file:DANRE/he1.2/he1.2-uniprot.txt Note=Binds 1 zinc ion per subunit. |
| GO:0006508 proteolysis | IDA PMID:19021768 Purification and characterization of zebrafish hatching enzy... | ACCEPT | Summary: ZFIN IDA from Sano et al. 2008, who purified ZHE1 and mapped six cleavage sites in the N-terminal regions of the egg-envelope subunit proteins ZP2 and ZP3; ZHE1 partially digests and swells the envelope, and cross-species digestion shows it cleaves the same sites in medaka envelope proteins as medaka HCE. Reason: The paper directly demonstrates proteolytic digestion of egg-envelope proteins by the purified zebrafish enzyme; he1.2 is the agent of the hydrolysis, so the process annotation is correct and core. Supporting Evidence: PMID:19021768 The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. PMID:19021768 partially digests the egg envelope and swells the envelope |
| GO:0008237 metallopeptidase activity | IDA PMID:19021768 Purification and characterization of zebrafish hatching enzy... | MODIFY | Summary: ZFIN IDA for the generic metallopeptidase term from the purification/characterization paper. The same paper shows the cleavage sites are internal to ZP2/ZP3, which makes the enzyme an endopeptidase, and UniProt assigns EC 3.4.24.67 (choriolysin H) on this evidence. Reason: The evidence is sound, but the term is the parent of metalloendopeptidase activity (GO:0004222), which the same evidence supports and which the gene already carries by IDA (PMID:20727360) and IBA. Replacing the parent with the child makes the ZFIN row consistent with the UniProt and PAINT rows; nothing in the annotation is denied. Proposed replacements: metalloendopeptidase activity Supporting Evidence: PMID:19021768 The six ZHE1-cleaving sites were located in the N-terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. file:DANRE/he1.2/he1.2-uniprot.txt FUNCTION: Metalloendopeptidase which participates in the breakdown of the egg envelope at the time of hatching. |
| GO:0035188 hatching | IDA PMID:19021768 Purification and characterization of zebrafish hatching enzy... | ACCEPT | Summary: ZFIN IDA for hatching (the emergence of the embryo from the chorion). Sano et al. 2008 show that ZHE1 is the enzyme purified from hatching liquid and that, unlike medaka's two-enzyme HCE/LCE system, zebrafish hatching is performed by this single enzyme, which digests the egg envelope. Reason: Participation test passed: the gene product performs the enzymatic step (digestion of the chorion) that constitutes hatching, rather than merely being required for a downstream outcome. It is the defining biological role of the protein and the origin of its name. Supporting Evidence: PMID:19021768 Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes file:DANRE/he1.2/he1.2-uniprot.txt FUNCTION: Metalloendopeptidase which participates in the breakdown of the egg envelope at the time of hatching. |
| GO:0005576 extracellular region | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT propagation of "is active in extracellular region" from the astacin node (PTN000013301), whose extant members (BMP1, meprins, tolloid, hatching enzymes) are secreted enzymes. For he1.2 the location is directly shown: the active enzyme is purified from hatching liquid (PMID:19021768) and it carries a signal peptide. Reason: The node placement is sound because secretion is a shared property of the astacin clade, and he1.2 has its own experimental support (the ZFIN entry appears in WITH/FROM as expected). The enzyme's substrate, the chorion, is extracellular, so this is also where it is active. Consistent with the EXP and IEA rows. Supporting Evidence: PMID:19021768 one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid file:DANRE/he1.2/he1.2-uniprot.txt SUBCELLULAR LOCATION: Secreted |
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Download this section (compressed HTML)Q: Is the propeptide of he1.2 removed autocatalytically or by another protease (Mmp13a has been proposed as an upstream activator), and which event triggers release of the active enzyme from hatching-gland granules?
Q: Do the two zebrafish paralogs he1.1 and he1.2 differ in expression or substrate specificity, or is he1.2 the only functionally relevant hatching enzyme, as the ZHE1/ZHE2 expression data suggest?
Experiment: CRISPR knockout of he1.2 (and the he1.1 paralog) followed by hatching assays and chorion ZP2/ZP3 cleavage analysis to test whether zebrafish hatching truly depends on a single enzyme.
Type: genetic knockout
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