Abstract
Although pairs of basic amino acids are common endoproteolytic sites in prohormones, the enzymes responsible for these cleavages have not yet been characterized. To investigate the specificity of these endoproteases, cDNAs encoding pro-neuropeptide Y (pro-NPY) containing all four pairs of basic amino acids were expressed in AtT-20 cells. Pro-NPY was selected as a model substrate because it undergoes a single cleavage at the sequence -Lys-Arg- during posttranslational processing. AtT-20 cells, a mouse anterior pituitary corticotrope line, were selected because they synthesize pro-adrenocorticotropic hormone (pro-ACTH)/endorphin and cleave a well characterized subset of the eight pairs of basic amino acids in the precursor. Altered cDNAs encoding pro-NPY with -Arg-Arg-, -Arg-Lys-, or Lys-Lys- at the cleavage site were used to generate stable cell lines. The production of NPY and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, reversed-phase high performance liquid chromatography, ion-exchange high performance liquid chromatography, tryptic peptide mapping, and microsequencing. Direct amino acid labeling confirmed the identity of the pair of basic amino acids at the cleavage site. Even when the four pairs of basic amino acids were presented in the same structural context, the rate, extent, and type of cleavage was substrate-specific. Pro-NPY(-Arg-Arg-) was cleaved at a rate similar to that observed for the wild-type pro-NPY(-Lys-Arg-). In contrast, pro-NPY(-Arg-Lys-) was cleaved at a much lower rate, and pro-NPY (-Lys-Lys-) was cleaved very poorly. Following endoproteolytic cleavage, the pair of basic amino acids present did not alter the production of mature NPY with a COOH-terminal Tyr-NH2. While two of the three mutant pro-NPY molecules were processed to wild-type carboxyl-terminal peptide, the carboxyl-terminal peptide derived from pro-NPY(-Arg-Lys-) contained an amino-terminal lysine residue, indicating that biosynthetic endoproteolysis occurred in the middle or at the amino terminus of the pair of basic amino acid residues at the cleavage site. Expression of wild-type or mutant pro-NPY inhibited cleavages within the endogenous pro-ACTH/endorphin; poorly cleaved pro-NPY mutants (Lys in the second position of the cleavage site) were the most potent inhibitors of pro-ACTH/endorphin cleavage.
Highlights
From the *Department of Neuroscience, The Johns Hopkins School of Medicine, Baltimore, §Department of Biochemistry, Purdue University, West Lufayette, Indiana 47907
We have examined the sequence preference of the endoprotease(s) in the secretory pathway of AtT-20 cells, using closely related mutant forms of pro-NPY as test substrates
TO attempt to explain the cleavage pattern observed in the endogenous pro-ACTH/endorphin in AtT-20 cells, one must deal with two major questions
Summary
HindIII-BarnHI restriction fragment from pS@GlO, a plasmid containing the human NPY cDNA (Minth et al, 1984), was cloned into complementary sites in MlSmplO. NPY-directed antibody, and the immunoprecipitate subjected to gel filtration as previously described (Dickerson et al., 1987). Cell extracts were immunoprecipitated with carboxyl-terminal peptide-directed antibody (Dickerson et al, 1987), subjected to gel filtration (Mains and Eipper, 1984), and digested with trypsin as above. Immunoprecipitates were subjected to gel chromatography as previously described (Dickerson et al, 1987) and fractions containing carboxyl-terminal peptide were pooled and dried under vacuum. Cell lines expressed pro-NPY at levels approximately 3-10% the level of proACTH/endorphin This level of expression was the maximal achieved for these last two mutants after eight separate transfections using calcium phosphate or lipofection, with over 75 drug-resistant lines screened for each of the R3sK3’ and K3’K3’ mutants. K3*K3’ cells was matched by a proportionally decreased level of pro-NPY mRNA compared to Mt.NPY/K38R39 cells, as determined by Northern analysis (Dickerson et al, 1987)
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