Are prohormones converted to hormones during secretion?

Medical Hypotheses15: 47-59,

1984

ARE PROHORMONESCONVEE?-I'EDTOHORMONES DURING SECRETICN? D.P.L.Green,Departmentof Pharmacology,Universityof Otago Medical School, P.O.Box 913, Dunedin,New Zealand.

A number of lines of evidencesuggest that the polypeptide prohormoneconvertingenzyme is a trypsin-likeserine protease. A model is proposed in which the convertingenzyme is activatedfrom an inactivezymogen during secretion. Convertingenzyme then activates co-secretedprohormoneproteolytically. An importantfeatureof the model lies in the geometryof the secretorygranule immediatelyafter exocytosis. It is suggestedthat initiallydiffusionof the granule contents is limitedenough to allow extensiveproteolysisto occur. Conversionof prohormoneto hormone is terminatedby diffusionof convertingenzyme and prohormonefrom the site of release. IRrRODucrION The biosynthesisof protein hormonessuch as insulin,glucagon, parathyroidhormone,etc. involvesthe synthesisof molecularforms larger than the polypeptideeventuallysecretedfrom the cell (1,2). Polypeptidehormonesare synthesizedby ribosomeson the cytoplasmic face of the endoplasmicreticulum. Nascent polypeptideis transferred to the lumen of the endoplasmicreticulumfrom where it proceedsto the Golgi apparatus. After segregationinto secretorygranules, polypeptideis dischargedfrom the cell by exocytosis. In coIRIK)n with other proteins synthesizedby polysomesassociatedwith membranesof the endoplasmicreticulum,"primarytranslationproducts"or "presecretory"proteinsare characterizedby the presenceof an amino terminalextensionwhich containspredominantlyhydrophobicamino acids. This "pre" sequence is removedby proteolyticcleavageshortly after synthesis (3,4,5). Removal of the "pre" sequenceleaves the hormonalpolypeptideas a propolypeptideor prohormone. This requires further proteolyticcleavageto generateactive hormone. 47 MH

(

Conversionof propolypeptideto active hormoneoccurs after transportto the Golgi region (6-12). The propolypeptideprecursors possess a slaw turnover,with an initialdelay of some 10-20 minutes post synthesis,after which cleavagecommences. Half-livesrange from about 20 minutes to one hour or longer (13,14). Some evidence suggeststhat the precursorsare activatedas they pass into the Colgi apparatusand/or into newly formed (pro)secretory granules (7,15-l&3) althoughequally there is evidencethat proalbuminis not convertedto albumin until close to or during secretion(19). The site of cleavageof the "pro" sequencesis usuallymarked by a pair of basic amino acids (Arg,Lys)(6,20-30). A small number of activationsites exist, however,in which one or both basic amino-acidsare replacedby non-basicresidues (31-36). IDENTIFICATIONOFTHEPF0HORM3NECONVEXTINGENZYMES Despite extensiveinvestigation, the identitiesof the enzymes which convertprohormoneto hormonehave yet to be established(1,2). A number of lines of evidencesuggest,hmever, that enzymes relatedto the digestiveenzymes trypsin,chymotrypsin,and carboxypeptidase are strong candidates. These include: i) the nature of the cleavagesites. In most cases this site is basic and would be susceptibleto cleavageby a trypsin-likeenzyme (6,20-32). Proinsulincan be readilycleavedby trypsinand carboxypeptidase A to reproducethe cleavagepattern in vivo (38) and trypsinconvertsa number of other propolypeptidesto active peptides (38-41). In the small number of cases in which cleavage occurs at other than basic sites, all but one are candidatesfor a chymotrypsin-like enzyme (33-35). ii) a human a -antitrypsinmutant, in which the specificityof the protease inhi .8. itor is shifted from being an anti-elastaseto an anti-trypsin,is associatedwith proalbuminaemia(42), suggestingthat the enzyme which activatespr~albumin is sensitiveto inhibitionby a serine protease inhibitor. Notwithstandingthis evidence,there are difficultieswith a trypsin-likeactivatingenzyme. Essentiallythese difficultiesarise because of the need to reconciletrypsin-likeactivity,which is normallyextracellular,with an intracellularactivationstep. Clearly, if a prohormoneis to be exposed to the activatingenzyme during passage through the cell the enzyme must not destroy the active hormone during the period of exposure. Either proteolysisis restrictedto the activationsite becausethe enzyme recognizesthat

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site and no other, or because access to other sites is restricted because of protein folding and/or stacking. There must be some suspicion that a trypsin-like protease would not be unduly restricted in its specificity and if that were the case, a hormone could only protect itself by burying sensitive sites. However, in the absence of hard data on the qpecificity of the activating enzyme this cannot be considered an over-riding objection to the intracellular involvement of trypsin-like proteases. A more substantial difficulty is the evidence that trypsin and other serine proteases are synthesized as zymcgens which require proteolytic activation, and that this activation normally occurs after secretion (43,44). Trypsinogen is initially activated by an extracellular protease, enterokinase. The trypsin produced then activates all three zymogens, trypsinogen, chymotrypsinogen, and procarboxypeptidase. Trypsinogen itself has some proteolytic activity and is capable of autoactivation , albeit at a low level when compared with that of trypsin (45). This basal activation of trypsinogen by trypsinogen is prevented from developing into rapid autoactivation by trypsin by the inclusion, within the granule, of a serine protease inhibitor (46). This inhibitor distinguishes between trypsinogen and trypsin and only binds to the latter. It exists in sufficient quantity to inhibit up to 25% of the potential trypsin activity, and given that only about 2-3% of secreted trypsinogen is present as trypsin, there is a wide safety margin. It is clear that this basic mechanism of trypsinogen activation must be modified if a trypsin-like protease is active within the secretory granule prior to secretion. Firstly, a mechanism must exist for activating the trypsin-like protease within the granule, or synthesizing the protease in a form which requires no proteolytic activation. Secondly, the active hormone must be immune to damage by the protease within the time-scale for passage of the granule through the cell. Thirdly, the secretory cell must be free of damage potentially caused by premature zymogen activation (44). It is against this background that cathepsin B has been suggested as a putative candidate for the converting enzyme (47-52). It has a substrate specificity similar to that of trypsin (53), is active at the pH which evidence suggests is present within secretory granules (54-56), and as a lysozomal enzyme, is capable of an intra-granular location. However, cathepsin B does not cleave either proPTH or glucagon correctly, and a general role for cathepsin B appears unlikely at this stage (57-59).

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AN ALTEEWATIVE MECHANISM FOR PFOHORBKNE ACTIVATION In the search for the converting enzyme, it has been assumed that the site of the activation step has been established beyond reasonable doubt: it is intracellular, at a site associated with the Golgi apparatus or the secretory granule (1). It follows that the enzyme is active within some sub-cellular compartment (and, in principle, extractable as active enzyme), is active at the pH within the compartment, and cleaves the prohormone at the appropriate cleavage sites but causes minimal additional damage. In the model proposed here, the emphasis is shifted substantially. It makes the assumption that the converting enzyme is a trypsin-like protease (which is stored as a zyrqen) and that such an enzyme cannot be active within the granule prior to secretion. In this, it follows closely the traditional pattern of zymogen storage. The converting enzyme is stored with the prohormone and is activated during secretion. Conversion of prohormone to hormone then follows. The starting point for this model is a group of cells, of which sperm and egg are the best characterized, in which activation of a trypsin-like zymogen takes place during secretion (60-64). Sperm and eggs are secretory cells in which secretion is a single discrete event. Synthesis of the secretory granules (acrosome, cortical granules) takes place before their discharge (65-69) and enables unequivocal identification of zymogen activation with the secretory event. Since the zymogens in these cells are activated in the absence of extracellular proteases, they represent self-activating systems. Moreover, zymogens are themselves activated by proteolysis. The mechanism by which this is achieved has yet to be established but one possibility is outlined below. The important point is that experimental evidence exists for autoactivation by proteolysis during secretion. It is a comparatively simple step to extend this proteolysis to include the activation of co-secreted zymogens, such as chymotrypsinogen and procarboxypeptidase, which may be involved in prohormone activation (33), as well as activation of co-secreted prohormones. The action of the converting enzyme(s) would be terminated by diffusion of substrate and enzyme from the site of release. Details of the self-activating generation of proteases in both sperm and egg remain substantially unknown. Firstly, it is not known whether enzyme activation occurs before or after fusion of the granule membrane with the plasma membrane, i.e. before or after exocytosis of the granule contents. At first sight, activation after exocytosis appears to be inherently unlikely, but this ignores both the geometry of release and the physical nature of the granule contents. In most cases, secretory granules are approximately spherical and undergo

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0

I

1

03 Fig.1. Diagramnaticrepresentationof secretion. (1) Secretory granule approachesplasma membrane. (2) Granule membranefuses Ath plasma membranewith concomitantexocytosisof granule contents. (3) Immediatelyfollowingexocytosis,granulevolume increasesdue to swellingof granule contents. Extracellularfluid is drawn into the granule space and outward diffusionof granule contents is retarded. The condensedgranule core begins to break up. It is during this phase that the model predictsactivationof both convertingenzyme and prohormonetakes place. (4) Granule contentsare lost from the surface of the secretorycell. Prohormoneconversionis terminatedby dilution.

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fusion at a single point (70). Even in sperm,where the secretory granule ultimatelyfuses with the plasma membraneover a wide area( there is evidencethat fusion is initiatedconsistentlyat only one or two sites (71). Immediatelyafter exocytosisthe secretorygranule contentsare retainedin a confinedspace with a restrictedoutlet for diffusionfrom the cell. Moreover,the physicalcondensationof granule contentswhich takes place in associationwith the Golgi (17) would furtherhinder diffusion. In addition,it is clear from the ultrastructureof secretionthat, in many cases, secretionis accompaniedby a rapid and substantialswellingof granule contents and thereforegranule volume (72-4). This is shown diagrammatically in Fig.1. The effect of this swellingwould be to draw extracellular fluid into the former granule before substantialdiffusionof granule contentstook place. The mechanismfor initiatingthe activationof proteolysisin sperm and egg is also unknown. It could be through the involvementof a second enzyme, itselfactivatedat secretion. Equally it could be the removalof an inhibitorof the trypsin-likeprotease,such as Zn2+ or H+ (54), or access by an activatingagent such as calcium is of the order of 1mM rhereas Ca2+. (Extracellular intragranularcalcium is likely to be close to the intracellularvalue of SO-1WrlM). The possibilityof controlby pH is attractivesince there is evidencethat the intra-granular pH is about 5 (54-56),and that autoactivationof trypsin-likeproteasesis suppressedat this PH. Exocytosistiouldexpose those contentsto the extracellularpH of about 7.4, at which pH autoactivation would take place. Whateverthe trigger for activation,the swellingof the granule contentsplays a potentiallyimportantrole in both alteringthe compositionof granule , and preventingoutwarddiffusionof fluid immediatelypost exocytosis the contents. Although it is envisagedthat the convertingenzyme is actually present in the secretorygranule togetherwith the prohormone,the model can be extendedto a situationin which the convertingenzyme is extracellular,but its own activatoris intragranular.Exocytosis, because it causes granule contentswelling,could draw a converting enzyme onto both its activatingenzyme and its substrate. This mechanismwould account,for example,for any involvementby plasmin in proinsulinconversion,where evidencesuggestsplasminogen activator (PA) may be co-secretedwith the prohormone(75). SOME CCNSEQUENCES OF THE MODEL --Undoubtedlythe most ilmportant predictionof the model is that the convertingenzymes are zymogenswhich are activatedon secretion. The

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enzyme(s)would be closely related to the establishedzymogens trypsin,chymotrypsinand carboxypeptidase.It followsthat any convertingenzymes extractedby cellulardisruptionwill not be detectableunless the conditionsof extractionfavour simultaneous activation. The model predictsthat, unless extractionconditions fortuitouslyprecipitateactivation,near non-existentlevels of active convertingenzyme will exist after disruption,and that the interferencefrom enzymes derived from other sub-cellularsources would make identificationof the convertingenzyme difficult. The most importantobjectionto the model lies in its precise identificationof the site of conversionas the cell boundary. This appears to run counter to existingexperimentalevidencewhich suggestsan intracellularsite (1,7,15-B). One importantpoint which has to be borne in mind is the questionof whether there is a single mechanismof prohormoneconversion. Assumingthat there is, however, then the evidence for an intracellularconversionsite must be treated with caution. Firstly,the evidence is not uniform,since the evidence is that proalbuminis convertedto albumin close to or during secretion (19). Secondly,the evidencefor an intracellularsite depends in part on the superpositionof kineticdata from the pancreaticendocrinecell on that of the pancreaticendocrinecell (l), a step which requiresfurtherexperimentaljustification.

coNcWsIoNs Prohormonesare currentlyconsideredto be convertedto hormones vrithinthe cell by a trypsin-likeprotease. Notwithstandingthe evidence in support of this view, the search for prohormoneconverting enzymes has so far proved inconclusive. In this paper, the experimentalevidencehas been re-examined. An alternativemechanism has been proposed in which prohormoneconversiontakes place during secretion. The model containsa number of predictionswhich can be tested experimentally. ACKNOWLEDGMENTS This work was supportedby the MedicalResearchCouncil of New Zealand.

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