Enterokinase, theinitiator ofintestinal digestion, isamosaic protease composed ofadistinctive assortment ofdomains

Enterokinase isaprotease oftheintestinal brushborder thatspecifically cleaves theacidic propeptide fromtrypsinogen toyield active trypsin. This cleavage initiates acascade ofproteolytic reactions leading totheactivation of manypancreatic zymogens. Thefull-length cDNAsequence for bovine enterokinase andpartial cDNAsequence forhuman enterokinase weredetermined. Thededuced aminoacidse- quences indicate that active two-chain enterokinase isderived fromasingle-chain precursor. Membrane association maybe mediated byapotential sigal-anchor sequence neartheamino terminus. Theaminoterminus ofbovine enterokinase also meetstheknownsequence requirements forprotein N-myris- toylation. Theamino-terminal heavy chain contains domains that arehomologous tosegments ofthelowdensity lipoprotein receptor, complement components COrandCls, themacro- phagescavenger receptor, anda recently described motif shared bythemetalloprotease meprin andtheXenopus AS neuronal recognition protein. Thecarboxyl-terminal light chain ishomologous tothetrypsin-like serine proteases. Thus, enterokinase isamosaic protein withacomplex evolutionary history. Theaminoacidsequence surrounding theamino terminus oftheenterokinase light chain isITPK-IVGG (hu- man)orVSPK-IVGG(bovine), suggesting thatsingle-chain enterokinase isactivated byanunidentified trypsin-like pro- tease that cleaves theindicated Lys-fle bond. Therefore, en- terokinase maynotbethe"first" enzymeoftheintestnal digestive hydrolase cascade. Thespecificity ofenterokinase for theDDDDK-Isequence oftrpsinogen maybeexplained by complementary basic-amino acid residues clustered inpoten- tial S2-S5 subsites. Allanimals needtodigest exogenous macromolecules with- outdestroying similar endogenous constituents. Theregula- tion ofdigestive enzymesis, therefore, afundamental re- quirement (1). Vertebrates havesolved this problem, inpart, byusing atwo-step enzymatic cascade toconvert pancreatic zymogens toactive enzymesinthelumenofthegut.The basic features ofthis cascade weredescribed in1899 byN.P. Schepovalnikov, working inthelaboratory ofI.P.Pavlov (2). Extracts oftheproximal small intestine wereshownto strikingly activate thelatent hydrolytic enzymes inpancre- atic fluid. Pavlov considered this intestinal factor tobean enzymethatactivated other enzymes, ora "ferment of ferments," andnamedit"enterokinase." Theimportance of this protease cascade isemphasized bythelife-threatening intestinal malabsorption thataccompanies congenital defi- ciency ofenterokinase (3,4). Enterokinase activates bovine trypsinogen bycleaving after thesequence VDDDDK,releasing anamino-terminal activation peptide (5, 6). Theacidic DDDDK sequence ofthe trypsinogen-activation peptide isconserved amongverte- brates (7), except forthesimilar sequences oftrypsinogens fromlungfish (IEEDKandLEDDK)andAfrican clawed frog (FDDDK). Enterokinase prefers substrates withthese- quence DDDDK,whereas thepresence ofaspartate residues markedly inhibits theability oftrypsin tocleave suchsub- strates (8). Forexample, toward bovine trypsinogen the catalytic efficiency ofenterokinase is12,000-fold (porcine) (9)or34,000-fold (bovine) (10) greater thanthat ofbovine trypsin. Thisreciprocal specificity protects trypsinogen against autoactivation bytrypsin andpromotes activation by enterokinase inthegut. Enterokinase hasbeenpurified fromporcine (11), bovine (10, 12,13), human(14), andostrich intestine (15). Withthe possible exception ofhumanenterokinase, which wassug- gested tobeaheterotrimer (14), enterokinase appears tobe adisulfide-linked heterodimer withaheavy chain of82-140 kDaandalight chain of35-62kDa.Mammalian enteroki- nases contain 30-50%o carbohydrate, which maycontribute to theapparent differences inpolypeptide masses. Theheavy chain ispostulated tomediate association with theintestinal brush border membrane (16), although nodirect evidence for this function hasbeenreported. Thelight chain contains the catalytic center. Basedonsusceptibility toinhibition by chemical modification oftheactive-site serine andhistidine residues (9-11, 17)andonthepartial aminoacid sequence (18) andcDNAsequence ofthebovine enterokinase light chain (19), enterokinase isamember ofthetrypsin-like family ofserine proteases. Enterokinase stands atornearthetopofaregulatory enzymecascade that successfully limits theactivity ofdiges- tive hydrolases tothegut,butthere isnostructural expla- nation forenterokinase membrane localization, substrate specificity, orexpression specifically intheproximal small intestine. Toaddress these questions wehavecharacterized cDNAclones forbovine andhumanenterokinase.§ MATERIALSANDMETHODS Materials. Purified calf enterokinase (EK-3, 131units/4g) wasfromBiozyme Laboratories (SanDiego). Fresh bovine tissues werefromalocal abattoir. AminoAcidSequencing. Enterokinase (16pg) wasreduced with0.5%(vol/vol) 2-mercaptoethanol, separated byelec- trophoresis (20), transferred toanImmobilon Pmembrane (Millipore) byelectroblotting, andstained withCoomassie brilliant blue. Theexcised light-chain band(=47kDa)was subjected toautomated Edmandegradation with anApplied Biosystems model470Asequencer (21)equipped witha model120Aphenylthiohydantoin analyzer.