The Role of the Membrane-spanning Domain of Type I Signal Peptidases in Substrate Cleavage Site Selection

Carlos, Joseph L.; Paetzel, Mark; Brubaker, Greg; Karla, Andrew; Ashwell, C. M.; Lively, Mark O.; Cao, Guoqing; Bullinger, Patrick R.; Dalbey, Ross E.

doi:10.1074/jbc.m007093200

Cited by 52 publications

(44 citation statements)

References 40 publications

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“…To investigate any Ag processing function of this protease, the two potential cleavage sites within the nNP-6K ricin were separately rendered noncleavable by replacing the Ϫ3 and Ϫ1 positions with arginine. As described previously (36), such changes would preclude processing by signal peptidase. Indeed, replacement of the preprolactin cleavage site with either RFRKQ or RGRTV, followed by in vitro translation in the presence of canine microsomal membrane, confirmed that such substitutions prevented cleavage (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation

Smith

Gallimore

Jones

et al. 2002

The Journal of Immunology

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In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8+ T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.

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Section: Resultsmentioning

confidence: 99%

Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation

Smith

Gallimore

Jones

et al. 2002

The Journal of Immunology

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“…The leader peptide is unlike those of other lantibiotics and resembles the signal peptides of proteins that are exported via the general secretory (Sec) pathway (for example, the three amino acid residues before the cleavage site of mature cinnamycin, AFA, conform to the AXA motif required for cleavage by type I signal peptidases; ref. 12).…”

Section: Resultsmentioning

confidence: 99%

Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005

Widdick

Dodd

Barraille

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

115

140

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Lantibiotics are ribosomally synthesized oligopeptide antibiotics that contain lanthionine bridges derived by the posttranslational modification of amino acid residues. Here, we describe the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005, the first, to our knowledge, lantibiotic gene cluster from a high G؉C bacterium to be cloned and sequenced. The cin cluster contains many genes not found in lantibiotic clusters from low G؉C Gram-positive bacteria, including a Streptomyces antibiotic regulatory protein regulatory gene, and lacks others found in such clusters, such as a LanT-type transporter and a LanP-type protease. Transfer of the cin cluster to Streptomyces lividans resulted in heterologous production of cinnamycin. Furthermore, modification of the cinnamycin structural gene (cinA) led to production of two naturally occurring lantibiotics, duramycin and duramycin B, closely resembling cinnamycin, whereas attempts to make a more widely diverged derivative, duramycin C, failed to generate biologically active material. These results provide a basis for future attempts to construct extensive libraries of cinnamycin variants.C innamycin (Fig. 1) is a peptide antibiotic produced by several Streptomyces strains, including Streptomyces cinnamoneus cinnamoneus DSM 40005. It is assumed to be made by posttranslational modification and processing of a larger ribosomally synthesized peptide. Modifications include the formation of lanthionine bridges, which are thio-ether bridges made when cysteine residues react with dehydroalanine or dehydrobutyrine moieties that have in turn been formed by dehydration of serine or threonine residues, respectively. The possession of lanthionine bridges defines cinnamycin as a lantibiotic. The lantibiotics studied so far are made as prepeptides, each consisting of a leader peptide and a propeptide. Modification of amino acid residues in the propeptide occurs before cleavage of the leader sequence, which releases the mature lantibiotic. In addition to lanthionine residues, lantibiotics may also contain other posttranslationally modified amino acid residues. Thus, cinnamycin contains a ␤-hydroxy-aspartate residue and a lysino-alanine bridge (apparently formed in a similar manner to a lanthionine bridge but with a lysine residue replacing cysteine; Fig. 1). Lantibiotic synthesis and classification have been reviewed (1).There are two major subclasses of lantibiotics. Type A lantibiotics have been studied intensively and have a tertiary structure that is relatively flexible and rod-like. In contrast, the less-studied type B lantibiotics have a relatively globular and inflexible tertiary structure (2). Whereas type A lantibiotics form pores in the cell membrane, type B lantibiotics may inhibit enzymes involved in cell-wall biosynthesis (3). Cinnamycin is closely related to type B lantibiotics duramycin, duramycin B, duramycin C, and ancovenin. These compounds are all derived from 19-aa propeptides and have lanthionine residues in similar position...

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“…pre-A2-AmyL), rather than to the structural features of sf-SipS-His (Bam). Dalbey and co-workers were recently able to show that even the intact SipS (Bsu), which was produced from a fusion with the maltose-binding protein (Carlos et al, 2000), was capable of efficient processing of a pro-OmpA nuclease A hybrid precursor in the absence of detergent (R. E. Dalbey, personal communication). This is the same precursor that was used to demonstrate the detergent-dependent activity of the soluble derivative of the E. coli SPase I (Tschantz et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Detergent-independent in vitro activity of a truncated Bacillus signal peptidase

et al. 2001

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The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory preproteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.

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The Role of the Membrane-spanning Domain of Type I Signal Peptidases in Substrate Cleavage Site Selection

Cited by 52 publications

References 40 publications

Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation

Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation

Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005

Detergent-independent in vitro activity of a truncated Bacillus signal peptidase

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