Endoproteolytic processing of recombinant proalbumin variants by the yeast Kex2 protease

Abstract

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.

Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2′ positions

“…3A, lane 2). This was despite the iacubations with proalbumin having a 33-fold higher enzy-] le : substrate ratio compared to the assays with the synthetic " able 2 l, inetic parameters for processing by Yap3 peptides, A number of natural proalbumin variants (-2Arg ~ His, -1Arg ~ Gln, -2Arg--* Cys, + 1Asp ~ Val) [17] and recombinant proalbumin variants (-4Val~Arg, -2Arg ~ Lys, -4Val ~ Arg-2Arg ~ His, + 1Asp ~ Arg) [10] were also tested as substrates for Yap3 (not shown). Yap3 did not release the propeptide from any of these variants.…”

“…The reactions were stopped after 16 h by the addition of pepstatin A to 50 gg/ml. Proalbumin was incubated with Kex2 as previously described [10]. Reactions were analysed in 1% agarose gels using Trisbarbital buffer pH 8.6 [14].…”

“…We have previously shown that the albumin propeptide RGVFRR is specifically cleaved from the N-terminal of proalbumin by Kex2 in vivo and in vitro [10][11][12]. In this study we investigate the specificity of Yap3 in vitro using a series of synthetic proalbumin peptides and natural and recombinant proalbumin variants.…”

Synthesis and Characterization of the First Potent Inhibitor of Yapsin 1

“…Extensive analyses of its specificity (15,(22)(23)(24) have revealed that substrates containing multiple basic residues surrounding the scissile bond are cleaved with high efficiencies. The nature of this specificity is not completely understood even though molecular modeling (Protein Data Bank code 1YPS) (24) has revealed that yapsin 1 has a more open active site compared with other aspartyl proteases and that many of its subsites are electronegative, thus allowing for the accommodation of positively charged residues within and surrounding the cleavage site (24).…”

Antibody fragments may be incorrectly processed in the yeast Pichia pastoris

“…Variants of the pre-pro-a-F with different amino acid changes in the vicinity of the pro-region processing site (Table 1) were designed on the basis of the earlier research (Bevan et al 1998;Ledgerwood et al 1995;Rholam et al 1995;Rockwell and Fuller 1998;Suzuki et al 2000). Corresponding coding DNA fragments were synthesized by PCR using Pfu DNA polymerase (Fermentas) and then cloned and sequenced on the pPH93 vector as in-frame fusions with the L-chain gene.…”