Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides

Abstract

The Bacilus subtilis ai-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis a-amylase signal peptides of different lengths and the mature thermostable a-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable at-amylases and by in vivo pulse-chase experiments. In B. subfilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and ca-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.Most exported proteins are synthesized as precursors with an NH2-terminal extension, the signal peptide, which is removed by a specific enzyme. The signal peptide and signal peptidase mechanisms for the secretion of exported proteins are similar in both prokaryotic and eukaryotic cells (2,8,10,22). In prokaryotic cells, the amino acid residues at positions -3 and -1 are important for cleavage site recognition by the signal peptidases when signal peptides are cleaved between positions -1 and + 1, with position -1 being particularly important (30). On the basis of the amino acid sequences of signal peptides reported by Watson (31) and Perlman and Halvorson (23), we compared the amino acid residues at positions -3 and -1 in 34 exported proteins of prokaryotic cells, excluding lipoproteins, whose signal peptides are cleaved by signal peptidase II (6). Eighty percent (27 of 34) of the amino acid residues at position -1 consisted of Ala, 44% (15 of 34) of the amino acid sequences at positions -3 and -1 were Ala-X-Ala, and 9% (3 of 34) of the sequences were Val-X-Ala and Thr-X-Ala. The sequence Ala-X-Ala predominates at positions -3 and -1 of the signal peptide cleavage site in precursors of exported proteins.Yamazaki et al. (34) predicted the amino acid sequence around the signal peptide cleavage site of Bacillus subtilis a-amylase to be Gly-27-Pro-Ala-Ala-Ala-Ser-Ala I GluThr-35. The cleavage site was confirmed as the peptide bond between Ala-33 and Glu-34 (Fig. la) In the signal peptide of AmyE, there were two Ala-X-Ala sequences at positions 29 to 31 and 31 to 33 near the signal peptide cleavage site. A cleavage s...

Study of mycoloyl transferase transport across the cell envelope of Corynebacterium glutamicum

“…The difference between the kinetics of processing and of the appearance of the protein in the cell wall indicates that signal peptide cleavage occurs early in the transport process and that a later step of PS1 transport is rate‐limiting. The rate of PS1 processing is similar to that determined for other Gram‐positive proteins such as PS2, the S‐layer protein of C. glutamicum (half‐life of 50 s, unpublished results), and α‐amylase from B. subtilis [21]. Therefore, the length of the signal sequence of PS1 which is quite long (43 aa versus 31 aa for most Gram‐positive bacteria) is not a critical parameter for an efficient cleavage by the leader peptidase.…”

Study of mycoloyl transferase transport across the cell envelope of Corynebacterium glutamicum

“…The di¡erence between the kinetics of processing and of the appearance of the protein in the cell wall indicates that signal peptide cleavage occurs early in the transport process and that a later step of PS1 transport is rate-limiting. The rate of PS1 processing is similar to that determined for other Gram-positive proteins such as PS2, the S-layer protein of C. glutamicum (half-life of 50 s, unpublished results), and K-amylase from B. subtilis [21]. Therefore, the length of the signal sequence of PS1 which is quite long (43 aa versus 31 aa for most Gram-positive bacteria) is not a critical parameter for an e¤cient cleavage by the leader peptidase.…”

Systematic Screening of All Signal Peptides from Bacillus subtilis: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria

“…Here, the effects were not reproduced even when beneficial modifications were transferred to other SPs fused to the same secretion partner. 1,2,22,23 Furthermore, secretion experiments in E. coli reported an influence of the N-terminal part of the mature protein in secretion efficiency, indicating the importance of an optimal interaction between signal peptide and secretion target. [24][25][26][27] The best SP for the secretion of one target protein is not automatically the best, or even a sufficient SP, for the secretion of another different target protein.…”