An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L-like cysteine proteases

Abstract

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

“…It can be seen that there is a rough reciprocal correlation between structural break down of the mutant P‐domain and the functional parameters. Pulse‐chase experiments in HEK‐cells with zymogens, bearing the same mutations, confirmed this trend (Kreusch et al 2000). This favors the hypothesis, based on physicochemical studies, that isolated propeptides, at least in complex with the corresponding enzymes, have the same fold as the proregions in the maternal zymogens (Maubach et al 1997; Jerala et al 1998; Ogino et al 1999).…”

“… Effect of scanning mutagenesis of highly conserved prosequence residues on human cathepsin S propeptide function; “XnpA” means that amino acid X in position n of the propeptide is substituted by Ala. Propeptide mutants have been expressed, purified, and characterized as published by Kreusch et al (2000). Renaturation rates ( left ordinate, •) are from Pietschmann et al (2002).…”

“…8). The same mutations in the zymogen affected neither targeting nor maturation and secretion in transfected HEK cells (Kreusch et al 2000). Although the P‐domain structure of these mutants remained intact, the inhibitory capacity was shown to be reduced by about one order of magnitude (Fig.…”

“…The complete sequence of human pre‐procathepsin S, with the only potential glycosylation site in the proregion at Asn89p (HCS1/4) (Wiederanders et al 1992), was cloned into the plasmid pcDNA3.1 (Invitrogen) as described previously (Kreusch et al 2000). The Cys25 → Ala mutation, introduced to prevent autocatalytic processing of the zymogen, was created by PCR‐based site‐directed mutagenesis according to the ExSite protocol (Stratagene) with the following oligonucleotide primers (the altered bases are underlined):…”

Early stages of seed development in Brassica napus: a seed coat‐specific cysteine proteinase associated with programmed cell death of the inner integument

“…The similarity of BnCysP1 to papain (39% identity) is evident throughout, suggesting that BnCysP1 is also synthesized with an N ‐terminal prepro region, as detailed below. A non‐contiguous ERFNIN motif in the pro‐region of papain family (Rawlings and Barrett, 1994), associated with obstructing an otherwise self‐destructive enzymatic activity (Kreusch et al ., 2000), is also conserved in BnCysP1 (Figure 2). The predicted mature peptide would start at Leu 127 to give a 217 aa polypeptide (≈23 kDa), and a Pro residue, required for blocking any N‐terminal proteolysis of the mature form (Rawlings and Barrett, 1994), is also present immediately after the Leu.…”