Abstract
Macrophages actively internalize macromolecules into endosomal vesicles containing proteases. The plant toxin, ricin A chain delivered into this pathway by receptor-mediated endocytosis, was found to be exquisitely sensitive to cleavage by these proteases. Proteolytic fragments of ricin A chain were generated within cells as early as 2-3 min after internalization. Toxin proteolysis was initiated in early endosomal vesicles, and transport to lysosomes was not required. As endosomes transit the cell, their lumenal pH drops from neutral to acidic. Previous studies in macrophages had suggested that endosomal proteolysis is dependent on vesicle acidification. Isolated endosomal vesicles containing ricin A chain catalyzed the cleavage of this protein in vitro; however, proteolysis was observed at both neutral and acidic pH. Experiments using isolated endosomes demonstrated that both cysteine and aspartyl proteases were responsible for the cleavage of ricin A chain. The cysteine protease, cathepsin B, catalyzed toxin proteolysis in endosomes between pH 4.5 and 7.0 while aspartyl protease activity was maximal below pH 5.5. Radiolabeling the lumenal contents of macrophage endosomes confirmed that both the cysteine protease, cathepsin B, and the aspartyl protease, cathepsin D, were present in these vesicles. These proteases were not present on the plasma membrane but were found in early endosomes indicating they are derived from an intracellular source. The presence of proteases with different pH optima in early endosomes suggests that processing in these vesicles may be regulated by changes in endosomal pH. This result represents an important difference in protein processing in endosomes versus lysosomes and provides new insights into the function of endosomal proteases.
Highlights
Macrophages actively internalize macromolecules proteolytic enzymes have been detected within endosomes, into endosomal vesicles containingproteasesT. he suggesting these vesicles serve as a site of intracellular plant toxin, ricin A chain delivered into this pathway processing
These studiessuggestedthat not present on the plasma membrane but were found the protease content of endosomes differs fromlysosomes, in early endosomes indicating they are derived from protein processing in each of these vacuolar compartments a n intracellular source
Two of the proteases, cathepsin B and the insulin-specific protease identified in endosomes, have broad pH optima and are catalytically active at neutralpH.These enzymes should be active in early endosomes where pH values between 6.5 and 7.4 have been the function of endosomal proteases
Summary
From the Departmentof Cell Biology and Physiology, Washington UniversitySchool of Medicine, St. The lumenal pH of these sviTencuneeolgsdeuxigsotci,renslaeosaltmnepaddtcreooisadtttthrecirafoiaaidnltnciysascepsit.niitosPodwrnorttaeh.stsvooeIcimsloeoiyluanllsla,siotttpsiehsaordetotmuietereddenloisudelwmyiosnssaeioissnnmeaniaalsoemrlpntldayHvdreceeoprdsqsoeiroucnpomildhpreeeaassdlngcv.tefoesrAonson-s-mhadval5icegtAasaiinvncdildltieymsi6,nidtt(pree7orrd)aop.Cctsnethuifiaornmondnmbgesee.nrsnateiounafrraeptnnrideoooutnestaroasmleostarolapuapHcnpifedomailcrdaivytnoagrleu,rgeeaussnilbdadeteetwripenercoeeetnpnetdaposoHre-taining ricin A chain catalyzed the cleavage of this somes, in contrast with lysosomes which contain a full comprotein in vitro; proteolysis was observed at plement of acid hydrolases, proteases, and peptidases. Ricin A chain cleavage at pHvalues above 6.0 These studies demonstrate that changes in endosomal pH play an important role in modulating proteolysis in thiscompartment.Both cathepsins D and B are found in lysosomes as well as endosomes, demonstrating that processing by these enzymes is.
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