THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Val. 266, No. 6, issue of February 25, pp. 3408-3410,1991 Printed in U.S.A.

A de Novo Designed Signal Peptide Cleavage Cassette Functions in Viuo*

(Received for publication, May 21,1990)

IngMarie Nibson and Gunnar von Heijne From The D e ~ r ~ ~ * t o ~ ~ o ~ c u ~ r ~ i o l o ~ ~ . Karol~nska Institute Center for B ~ ~ e h ~ ~ g y , NOVUM, ” I

S-14152 ~ ~ d ~ * g e , Sweden

Leader peptidase (Lep) is a membrane-bound enzyme of the ~ s c h e ~ ~ c h ~ ~ coli inner membrane that serves to remove signal peptides from exported proteins. Statis- tical and experimental studies of known signal peptides have defined a short C-terminal region that seems to provide the information for correct cleavage by Lep. Based on the patterns of conserved amino acids found in this region, we have designed a signal peptide “cleavage cassette.” This cassette is processed at the expected site when introduced after an uncleaved sig- nal peptide. Furthermore, processing is blocked in the predicted manner when the f-3,-1)-rule for signal peptide cleavage is violated. This suggests that current understanding of the sequence requirements for signal peptide cleavage is sufficiently advanced to be used in, e.g. protein engineering applications.

Signal peptides on secretory proteins produced in both prokaryotic and eukaryotic cells have a conserved overall design (1): an N-terminal, positively charged region (n-re- gion), a central hydrophobic region (h-region), and a slightIy polar C-terminal region (c-region) that contains the cleavage site recognized by the signal peptidase enzyme (called leader peptidase, Lep, in Escherichia coli (2)). The c-region is often delimited from the h-region by one or two helix-breaking residues, and its C-terminal end is characterized by the so- called (-3,-l)-rule (3), ie. only small, nonpolar amino acids are found in positions -3 and -1 counting from the cleavage- site.

The (-3,-l)-rule has been thoroughly tested in both pro- karyotic and eukaryotic systems (4, 5). In general, residues not found in these positions in known signal peptides block cleavage when introduced into otherwise wild type sequences. The role of the helix-breaking residues has also been studied; again, their presence seems to promote efficient cleavage, although they do not seem to be absolutely required (6, 7).

Encouraged by these results, we wanted to try the comple- mentary approach: to start from an uncleaved signal peptide and turn it into a cleavable one by the introduction of a “cleavage cassette” designed according to the positional amino acid preferences in the e-region. We now report that such a cassette can indeed be constructed and that it functions to promote efficient cleavage at the intended site in two different contexts, thus suggesting that engineering of signal peptidase

* This work was supported by grants from the Swedish Natural Sciences Research Council and the Swedish National Board for Technical Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

cleavage sites into otherwise uncleaved signal peptides is straightfo~ard.

EXPERIMENTAL PROCEDURES

M a ~ e r i a ~ - T ~ s i n , soybean trypsin inhibitor, chicken egg white lysozyme, and phenylmethylsulfonyl fluoride were from Sigma. All enzymes were from Promega.

from the PING1 plasmid (8) in E. coli strain MC1061 (9). Strains and Plasmids-Leader peptidase mutants were expressed

DNA Teehn~~s-Si te -spec i~c mutagenesis was performed ac- cording to the method of Kunkel (lo), as modified by Geisselsoder et ai. (11). All mutants were confirmed by DNA sequencing of single- stranded M13 DNA using T7 DNA polymerase (Pharmacia). Cloning into the PING1 plasmid was performed as described in Ref. 12.

Assay of Membrane Topology-E. coli strains transformed with the PING1 vector carrying mutant leader peptidase (lep) genes under control of the arabinose promoter were grown at 37 “C in M9 minimal medium supplemented with 100 pg/ml ampicillin, 0.5% fructose, and all amino acids (50 pg/ml each) except methionine. Overnight cultures were diluted 1:35 in 1 ml of fresh medium, shaken for 3.5 h at 37 “C, induced with arabinose (0.2%) for 5 min, and labeled with [%) methionine (150 pCi/ml). After 1 min, nonradioactive methionine was added (500 pg/ml) and incubation was stopped by acid precipi- tation (trichloroacetic acid, 10% final concentration) or by chilling on ice for protease accessibility studies. In the latter case, the cells were spun at 15,000 rpm for 1 min, resuspended in ice-cold buffer (40% w/v sucrose, 33 mM Tris, pH 8.0), and incubated with lysozyme (5 pg/ml) and 1 mM EDTA for 15 min on ice. Aliquots of the cell suspension were incubated 1 h on ice, either with 0.75 mg/ml trypsin or with 0.75 mg/ml trypsin + 0.8 mg/ml trypsin inhibitor + 0.33 mg/ ml phenylmethylsulfonyl fluoride. After addition of trypsin inhibitor and phenylmethylsulfonyl fluoride, samples were acid precipitated, resuspended in 10 mM Tris, 2% SDS,’ immunoprecipitated (13) with antisera to Lep, OmpA, and AraB, washed, and analyzed by SDS- PAGE and fluorgraphy.

Radiosequencing-MC1061 transformed with the appropriate plas- mid was grown overnight a t 37 “C in the same medium as above, diluted 1:35 into 0.5 mi of medium, shaken for 3.5 h at 37 “C, induced for 5 min with 0.2% arabinose, labeled for 10 min with 150 pCi of [%]Met, acid and immunoprecipitated with Lep antiserum as above, and purified by SDS-PAGE. The wet gel was exposed overnight, and the radioactive band was cut out, extracted with 0.1% SDS by gentle shaking overnight at 37 “C, concentrated 10 X by lyophilization, and sequenced on an Applied Biosystems 470A protein sequenator.

RESULTS AND DISCUSSION

Engineering of a Leader Peptidase Ckeavage Cassette-Our original design was based on the positional amino acid fre- quencies in the c-regions of signal peptides from E. coli (14). Thus 3 helix-breaking residues, Ser-Pro-Ser, were chosen to provide a clear point of transition from the apolar and most likely helical (15) h-region, followed by a (-3,-1)-pattern Ala-Gln-Ala. In addition, an additional alanine intended to become the N terminus of the cleaved product was put at the end of the cassette; Ala is very frequent in the +1 position of

’ The abbreviation used is: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

3408

Signal Peptide Cleavage Cassette 3409

secreted E. coli proteins (14). The Gln in position -2 was included so as to prevent cleavage between positions +1 and +2 (Gln has never been found in position -3 in signal peptides from E. coli).

This casette was introduced immediately downstream of the internal uncleaved signal sequence H2 in a derivative of leader peptidase which has a short insert and lacks residues 30-52 in the P1 domain (mutant Lep' (16)), Fig. 1, but is otherwise similar to the wild type molecule with both the N and C terminus facing the periplasm (16, 17) and in terms of its catalytic activity (18). When mutant H2-CC containing the casette is expressed from an arabinose-inducible plasmid, two forms of the protein can be immunoprecipitated by Lep antiserum (Fig. 2, lune 6). The large form is of the molecular weight expected for the intact protein, whereas the size of small dominating form is consistent with processing in or near the cleavage cassette.

To further characterize these different forms, a protease sensitivity assay (19) was used to determine their cytoplasmic or periplasmic location. The results presented in Fig. 3 show that the largest form is completely insensitive to prot,ease, indicating that it has not been translocated across the cyto- plasmic membrane, whereas the smallest form is readily di- gested, as expected for a periplasmic protein.

H2-CC

.... WLETGASVFPVLAIVLIVSPSAQAI-ARSFIYEPFQ ..,.

H I -CC

M A N M K K K K F A L I L V I A T L V T ~ I L W C V D K .... I

++/' ' N cytoplasm

FIG. 1. Position and sequence (underlined) of the signal peptide cleavage cassette in the two Lep-derived constructs H2-CC and HI-CC discussed in the test. Arrorr9s indicate the sites where the cleavage cassette is introduced. The Thr"' - Leu and the (;In and Tyr suhstitutions in the cleavage cassette in mutant H1-CC are indicated.

a b c

36 - 31 . B

D

24 .

FIG. 2. The H2-CC (lane b) and H1-CC (lane c ) constructs are processed to lower molecular weight forms of sizes con- sistent with cleavage in or near the cleavage cassette. T h e molecular mass standnrds (Innr a ) are wild type 1,ep (36 k l h ) , Lep A4-50 (31 k I h ) (12), and Lep pRD9 (9) with residues 62-76 deleted (24 kDa). Rased on t,heir amino arid compositions, the cleaved prod- ucts of HZ-CC and HI-CC have expected molerular masses of 28 and 32 kDa, respectively. Cells were lahcled hv [,""Sjmethionine for 2 min and proteins were immunoprecipitated hv Lep antiserum.

OmpA. .ep (uncleaved)

.ep (cleaved)

\raB lrVpain - + - +

FIG. 3. The cleaved form of the HP-CC mutant is sensitive to externally added protease and thus located in the peri- plasm. Protease sensitivitv o f OmpA shows that thr outer memtmnr is properly permeahilized to allow the protease arress to the pcri- plasmic space; protease resistance of AraH demonstrates that the inner membrane has been lrft intact antl prevents protease from entering the cytoplasm. /M/f lo riflhf: immunoprrripitation of OmpA from nontrypsinized (lonr 1 ) and trypsinizetl (lonr 2) cells: immrr. noprecipitation of 1,ep and AraH from nontrypsinized (Innr, 3 ) and tr-ypsinized (Innr 4 ) cells.

a b c d e f g h i

uncleaved- &"-==-.- - -

0 . 1 0.3 y.2

FIG. 4. The cleavage cassette promotes processing to a lower molecular weight form, and procwsing is inhihited by point mutations violnting the (-3,-l)-rule. Imnr n. cleavage cassette introtlured aftt,r HZ (mutant H2-C'C). /.an<, h. c l ~ a v a g ~ ras- sette introducedafter HI (mutant Hl-CY'). Imnrr, HI-C'C with Thr'' +IRU suhstitution (mutant HI-C(: TJ,). /nnr . sd - / , mutant H I - ( ' ( ? T,,L with (;In in positions -1 ( d ) , -2 ( r ) . antl ":1 (/) r o r r n t i n ~ from the predicted cleavage site (c . / . Fig. 1 ). ImnrsE-i, mutant HI-('(' T.-,l, with Tvr in positions -1 la), -2 t h ) , and -3 ( i ) .

300

250

200

k 150 I

i?

5 100

50

0 0 5 10 15 70 75

Cycle

FIG. 5. ["S]Met radiosequencing of the cleaved product oh- tained with the H2-CC mutant. c.l. Fig. 1. ' h seqm=nrr shown on top starts with the Ala residue predicted to tw immediately ('- terminal to the cleavage site. The 2 mrthioninrs in positions I ( i and 17 are undprlinrd.

We also introduced a variant of the cleavage cassette he- tween codons 20 and 21 in a derivative of leader peptidase which has been engineered such that the first hydrophohic region functions as an uncleaved signal peptide (mutant 4K,AB0-52,A62-76 (19) which has 4 extra N-terminal Ivsines and lacks residues 30-52 in the P I region, as well as the second transmemhrane domain H2, residues fi2-76), Fig. 1. In this case (mutant HI - (X) , the sequence of the cassette was Ser-Pro-Ser-Ala-His-AlalAla-Asp, with the His in position -2 intended to prevent cleavage of the Ala-Asp hond (S), and with the Asp included to match the known preference for acidic residues in position +2 in E. ml i signal peptides (20). Again, we observe efficient conversion to a processed form of a molecular weight expected for cleavage within the cassette (Fig. 2, l a m c) . The cleaved form is protease sensitive, sug- gesting a periplasmic location (not shown).

The persistence of a considerahle amount of full-length.

3410 Signal Peptide Cleavage Cassette

protease-resistant precursor suggested that the uncleaved sig- nal peptide may have been rendered less efficient as a trans- location signal by the cassette insertion, possibly because its hydrophobic region had been shortened. We thus sought to increase translocation efficiency by making the h-region more hydrophobic. Indeed, when ThrZ0 was replaced by Leu (mutant H1-CC TZoL), the fraction of processed molecules after a 2‘ labeling with [35S]Met was significantly increased (Fig. 4, lane c). The remaining full-length form is slowly translocated during a 10’ chase in both the H1-CC and the H1-CC T20L mutants (not shown).

The Cleavage Cassette Is Cleaved by Leader Peptidase-To ascertain that the cleavage cassette was indeed cleaved by leader peptidase, we performed two kinds of tests. First, two conveniently located methionines downstream of the internal signal sequence HZ allowed radiosequencing of the processed form of the HZ-CC mutant. Strong labeling was found in cycles 16 and 17, Fig. 5, demonstrating that the protein had been cleaved at the expected site.

Second, a series of point mutations designed to perturb the essential (-3,-1)-pattern were made in the H1-CC TzoL con- struct. Thus, positions -1, -2, and -3 were systematically replaced with Gln (Fig. 4, lanes d-f). Processing was blocked in the Ala” + Gln mutant and partially blocked in the A-3 + Gln mutant, but was unaffected in the His-’ + Gln mutant. The result with GlnP was somewhat unexpected, since Gln has never been found in this position in wild type bacterial signal peptides; however, in a recent study of the processing of MalE signal peptide mutations, a similar Ala-3 --* Asn replacement was also found to only partially block processing ( 5 ) . Our results thus suggest that both Asn and Gln when present in position -3 impair but do not completely block processing.

To further strengthen the case for leader peptidase-cata- lyzed cleavage, we made a similar series of mutants with Tyr rather than Gln in positions -1 to -3 (Fig. 4, lanes g - i ) . In this case, Tyr-3 blocked cleavage completely, as was also found for the MalE signal peptide ( 5 ) . Similarly, Tyr” blocked cleavage, but Tyr-’ had no effect. From these results, we conclude that the cleavage cassette was accurately cleaved by leader peptidase in both contexts.

This study demonstrates that one and the same c-region can promote context independent cleavage by leader peptidase irrespective of the precise sequence of the preceding h-region. Furthermore, an internal signal sequence can be cleaved by leader peptidase even though it is preceded by an N-terminal transmembrane region, Fig. 1. This has been shown previously

to be possible in eukaryotes (21); evidently it can also happen in bacteria. Finally, our results also underline the modular design of signal peptides: a transmembrane segment (HI) that does not function as a translocation signal in its normal contest can both be turned into an efficient uncleaved signal peptide by addition of a positively charged n-region (19, 22), and further be converted to a cleavable signal sequence by the introduction of a suitable c-region cleavage cassette.

Acknowledgments-Antisera were gifts from Dr. Ross Dalbey, Ohio State University (AraB), and from Dr. Ulf Henning, Tubingen (OmpA). Purified Lep used to produce Lep antiserum in rabbits was a gift from Dr. Bill Wickner, UCLA. Oligonucleotide synthesis and radiosequencing were done by Anne Peters and Maarit Narva at the Karolinska Institute Center for Biotechnology.

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