Disparate intracellular processing of human IL-12 preprotein subunits: Atypical processing of the P35 signal peptide

IL-12 is a heterodimeric cytokine produced by APC that critically regulates cell-mediated immunity. Because of its crucial function during immune resp onses, IL-12 production is stringently regulated, in part through transcrip tional control of its p35 subunit, which requires the differentiative effec ts of IFN-gamma for expression, To determine whether post-transcriptional a spects of IL-12 production might be regulated, we examined intracellular pr otein processing of each subunit, We report here that p40 and p35 subunits are processed by disparate pathways. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with transl ocation into the endoplasmic reticulum (ER), processing of p35 does not. Tr anslocation of the p35 preprotein into the ER was not accompanied by cleava ge of the signal peptide; rather, removal of the p35 signal peptide occurre d via two sequential cleavages. The first cleavage took place within the ER , and the cleavage site localized to the middle of the hydrophobic region o f the signal peptide, Although the preprotein was glycosylated upon entry i nto the ER, its glycosylation status did not affect primary cleavage. Subse quently, the remaining portion of the p35 signal peptide was removed by a s econd cleavage, possibly involving a metalloprotease, concomitant with addi tional glycosylation and secretion. Secretion could be inhibited by mutatio n of the second cleavage site or by inhibition of glycosylation,vith tunica mycin, In contrast, p40 secretion was not affected by inhibition of glycosy lation. Our findings demonstrate that IL-12 subunits are processed by dispa rate pathways and suggest new modalities for regulation of IL-12 production .