Endoproteolytic processing of recombinant proalbumin variants by the yeast Kex2 protease.
The yeast Kex2 protease is regarded as the prototype of the eukaryotic family of subtilisin-like serine proteases involved in processing after dibasic amino acid sequences. Here we investigate the specificity of Kex2 using recombinant human proalbumin variants. Proalbumins with the processing site sequences Arg-Arg and Lys-Arg were cleaved after the dibasic sequence at approximately the same rate by Kex2 in vitro, and yeast expressing either of these sequences secreted mature albumin into the culture medium. As expected, the Arg-Gly-Val-Phe-His-Arg-albumin (proalbumin Lille) was not a substrate for Kex2 and neither was the Arg-Gly-Arg-Phe-His-Arg-albumin. In contrast to the mammalian endoproteases furin and the hepatic proalbumin convertase, the Kex2 protease was adversely affected by a P4 arginine. There was an 85% decrease in the cleavage of Arg-Gly-Arg-Phe-Arg-Arg-albumin compared with normal; also chicken proalbumin with an Arg-Phe-Ala-Arg processing site sequence was not a substrate for Kex2. A P1' arginine had a marked negative effect on processing and N-terminal sequence analysis confirmed that cleavage was occurring at the P1-P1' bond. The sequence context surrounding the classical dibasic site is critical in determining susceptibility to cleavage by the Kex2 protease.
Ledgerwood
E C
EC
Molecular Pathology Laboratory, Christchurch Hospital, New Zealand.
George
P M
PM
Peach
R J
RJ
Brennan
S O
SO
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011228:Prealbumin / Q000378:metabolism*; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism*
D017931:DNA Primers; D011228:Prealbumin; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae