Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development☆

摘要

Thehatchingenzymeofmedakaconsistsoftwotypesofproteases(HCE,LCE).cDNAclonesforLCEandHCEwereisolatedfroma??gt11cDNAlibraryconstructedwithpoly(A)+RNAofDay3embryos.LCEcDNAis936bplongandcontainsan813-bpopenreadingframeencodingapreproenzymewitha20-amino-acidsignalsequence,a51-amino-acidpropeptide,anda200-amino-acidmatureenzyme.ForHCE,twodistinctcDNAs(HCE21,HCE23)havingnucleotidesequenceswith92.8%similaritywereobtained.ThesecDNAscontainopenreadingframesencodingpreproenzymesof279and270aminoacids,respectively.Thematureenzymeformsofbothconsistof200aminoacids,thesimilaritybetweenthembeing95.5%.OnNorthernblottinganalysis,thetranscriptsofLCEandHCEgeneswerefirstdetectedcoincidentallyinDay2embryosshortlybeforetheproductionofLCEandHCE,accumulatedthereafterinparallel,anddramaticallydecreasedafterhatching.Theaminoacidsequence,theHExxHmotif,whichisknowntoconstituteanactivesiteinsomeZnproteases,isalsofoundinLCEandHCE.However,thesequenceanalysesstronglysuggestthatboththeenzymesbelongtotheastacin(protease)family,beingdistinctfromseaurchinhatchingenzyme,whichisreportedlysimilartocollagenase.