Design of a transferrin-proteinase inhibitor conjugate to probe for activecysteine proteinases in endosomes

A new technique has been developed to identify active proteinases in endoso mes that does not require prior isolation of organelles and extraction of t he active enzymes. [I-125]Iodotyrosylalanyldiazomethane was reversibly conj ugated to transferrin to selectively deliver it to endosomes. The protein w as conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3 -(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enz ymes were separated from the transferrin after SDS/PAGE under reducing cond itions. Uptake of the protein-inhibitor conjugate and incorporation of inhi bitor into cathepsins was blocked at 4 degrees C, demonstrating that the co njugate enters cells by receptor-mediated endocytosis. Furthermore, endocyt osed transferrin-inhibitor conjugate could be recycled back to the extracel lular medium and binding to the transferrin receptor could be: blocked by n ative transferrin. Labelling of the enzymes was not blocked by incubating c ells at 16 degrees C, consistent with the majority of the reagent being tar geted to endosomes. The inhibited enzymes remained conjugated to transferri n, showing that the disulphide bond between the transferrin and inhibitor w as not reduced in the endosome. Results from these studies show that endoso mes contain both intermediate and late biosynthetic forms of active catheps in B, which are indistinguishable from those found in mature lysosomes. The se results indicate that the active enzymes in endosomes are not early bios ynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.