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To expand the structural understanding of gastropod hemocyanins and their physiological implications, here we report the single\u2010particle cryogenic electron microscopy (cryo\u2010EM) structure of\n                    <jats:italic>Pomacea canaliculata<\/jats:italic>\n                    snail hemocyanin (PcH) at 4.4\u2009\u00c5 resolution along with its intrinsic and proteolytically induced PO activity. PcH shows a\n                    <jats:italic>Megathura crenulata<\/jats:italic>\n                    (giant keyhole limpet) hemocyanin (KLH)\u2010type structure with a cylindrical shape, comprising 20 protomers assembled as di\u2010pentamers of antiparallel asymmetric dimers, organized in a D5 symmetry. Each protomer comprises eight paralogous functional units (FUs) sharing conserved structural features typical of hemocyanins. The achieved map resolution allowed delineation of interaction networks among adjacent subunits across quaternary structure tiers\u2014dimer, pentamer of dimers (decamer), and di\u2010pentamer of dimers (di\u2010decamer). Additionally, we identified six\n                    <jats:italic>N<\/jats:italic>\n                    \u2010glycosylation sites per protomer, totaling 120 glycan trees in the overall structure. Kinetic analysis of intrinsic PO activity using catechol as substrate revealed a Michaelis constant (\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>M<\/jats:sub>\n                    ) of 45.3\u2009m\n                    <jats:sc>m<\/jats:sc>\n                    and a catalytic rate constant (\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>cat<\/jats:sub>\n                    ) of 2.87\u2009min\n                    <jats:sup>\u22121<\/jats:sup>\n                    . This specific activity was enhanced by limited proteolysis using digestive and bacterial proteases. Potential protease cleavage sites were identified\n                    <jats:italic>in silico<\/jats:italic>\n                    , mapped onto the PcH model, and their accessibility assessed. Combined with molecular dynamics simulations, these findings suggest a structural basis for the PO induction mechanism. This study expands our knowledge of KLH\u2010type hemocyanins and provides clues into their PO activation, which is triggered by endogenous and\/or pathogen\u2010associated proteases. This further underscores the role of molluscan hemocyanins in the innate immune system.\n                  <\/jats:p>","DOI":"10.1111\/febs.70378","type":"journal-article","created":{"date-parts":[[2025,12,27]],"date-time":"2025-12-27T04:47:20Z","timestamp":1766810840000},"page":"2708-2731","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cryo\u2010\n                    <scp>EM<\/scp>\n                    structure of\n                    <i>N<\/i>\n                    \u2010glycosylated\n                    <i>Pomacea canaliculata<\/i>\n                    hemocyanin provides insights into its role in the immune response of gastropods"],"prefix":"10.1111","volume":"293","author":[{"given":"Tabata Romina","family":"Brola","sequence":"first","affiliation":[{"name":"Instituto de Investigaciones Bioqu\u00edmicas de La Plata \u201cProf. Dr. Rodolfo R. 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Brenner\u201d (INIBIOLP), Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET) Universidad Nacional de La Plata (UNLP)  Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guillermo Ignacio","family":"Ben\u00edtez","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Bioqu\u00edmicas de La Plata \u201cProf. Dr. Rodolfo R. Brenner\u201d (INIBIOLP), Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET) Universidad Nacional de La Plata (UNLP)  Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcos","family":"Gragera","sequence":"additional","affiliation":[{"name":"Centro Nacional de Biotecnolog\u00eda (CNB) Consejo Superior de Investigaciones Cient\u00edficas (CSIC)  Madrid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jose\u2010Maria","family":"Carazo","sequence":"additional","affiliation":[{"name":"Centro Nacional de Biotecnolog\u00eda (CNB) Consejo Superior de Investigaciones Cient\u00edficas (CSIC)  Madrid Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3379-0216","authenticated-orcid":false,"given":"Horacio","family":"Heras","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Bioqu\u00edmicas de La Plata \u201cProf. Dr. Rodolfo R. 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