Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.
Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.
Diefenbach-Jagger
H
H
St Vincent's Institute of Medical Research, St Vincent's Hospital, Fitzroy, Victoria, Australia.
Brenner
C
C
Kemp
B E
BE
Baron
W
W
McLean
J
J
Martin
T J
TJ
Moseley
J M
JM
D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein; D010957:Plasmids; D043484:Proprotein Convertases; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*
D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae