Effect of chicken lysozyme signal peptide alterations on secretion of human lysozyme in Saccharomyces cerevisiae

YOSHINORI TSUCHIYA'

Bioengineering Research Laboratory, National Institute of Animal Health, Kannondai 3-1-1, Tsukuba, Ibaraki 305, Japan

AND

HIROFUMI FUJISAWA, KEN-ICHI NAKAYAMA, HITOSHI NAGAHORA, AND YOSHIFUMI JIGAMI Molecular Biology Department, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305, Japan

Received May 1, 1993

TSUCHIYA, Y., FUJISAWA, H., NAKAYAMA, K., NAGAHORA, H., and JIGAMI, Y. 1993. Effect of chicken lysozyme signal peptide alterations on secretion of human lysozyme in Saccharomyces cerevisiae. Biochem. Cell Biol. 71: 401-405.

To investigate the structural requirements and functions of the N-terminal and the C-terminal regions of the chicken lysozyme signal peptide, the amino acids of each region were altered. The replacement of Gly(- 1) and Leu(-2) with Pro(- 1) and Ala(- 2) or Val(- 2), respectively, resulted in the complete shift of the cleavage site from position - 1 to - 2 in yeast (Saccharomyces cerevisiae). This shows that the introduction of a turn-promoting residue like Pro makes it possible to control the cleavage site of the signal peptide. Deletion of the positive charge and introduction of a negative charge in the N-terminal region decreased the lytic activity of secreted human lysozyme (HLY) and processing efficiency of preHLY, but the length and additional positive charge in this region had little influence. This suggests that the length of the N-terminal region scarcely influences the function of the signal peptide and that this region possibly interacts with the endoplasmic reticulum membrane to initiate the translocation of preprotein, similar to prokaryotic signal peptide. However, it needs only minimum positive charge for its function.

Key words: N-terminal region, signal peptide, cleavage site, human lysozyme, yeast.

TSUCHIYA, Y., FUJISAWA, H., NAKAYAMA, K., NAGAHORA, H., et JIGAMI, Y. 1993. Effect of chicken lysozyme signal peptide alterations on secretion of human lysozyme in Saccharomyces cerevisiae. Biochem. Cell Biol. 71 : 401-405.

Afin d'etudier les structures requises et les fonctions des domaines N-terminal et C-terminal du peptide de signalisation du lysozyme de poulet, les acides amines de chacun de ces domaines ont kt6 modifies. Le remplacement de la Gly(- 1) et de la Leu(- 2) respectivement par la Pro(- 1) et par la Ala(- 2) ou la Val(-2) entraine un dtplacement complet du site d'hydrolyse de la position - 1 a la position - 2 dans la levure (Saccharomyces cerevisiae). Cela demontre que I'introduction d'un acide amine engendrant un repliement, telle la proline, permet de contr6ler le site d'hydrolyse du peptide de signalisation. L'activite lytique du lysozyme humain skcrete et l'efficacite de transformation du prolysozyme humain sont diminuees B la suite de la deletion de la charge positive et I'introduction d'une charge negative dans le domaine N-terminal; cependant, la longueur et une charge positive additionnelle dans ce domaine ont peu d'effet. Cela suggtre que la longueur du domaine N-terminal influence peu la fonction du peptide de signalisation et que ce domaine peut se lier a la membrane du rtticulum endoplasmique et entrainer la translocation de la proenzyme, comme le peptide de signalisation des procaryotes. Cependant, seulement une charge positive minimale est necessaire a la fonction du peptide de signalisation.

Mots clds : domaine N-terminal, peptide de signalisation, site d'hydrolyse, lysozyme humain, levure. [Traduit par la redaction]

Introduction Signal sequences play an important role in the transloca-

tion of newly synthesized proteins across the membrane of the endoplasmic reticulum (eukaryotic cells) or across the cell membrane (prokaryotic cells). These sequences are com- posed of three structurally and functionally distinct regions: a positively charged N-terminal region, a central hydropho- bic core region, and a C-terminal region recognized by signal peptidase (von Heijne 1985). Statistical approaches to elu- cidate the structural and functional relationships of the sig- nal sequences have shown that only residues with small neutral side chains such as Ala, Gly, Ser, and Thr are usually found at the - 1 and - 3 positions (von Heijne 1983, 1986). Systematic site-directed mutagenesis of signal sequences has confirmed the importance of the physico-chemical properties of the - 1 and - 3 residues of the C-terminal region in prokaryotes (Borchert and Nagarajan 1991; Laforet and Kendall 1991) and eukaryotes (Folz et al. 1988). On the other

ABBREVIATIONS: HLY, human lysozyrne; IgG, immunoglobulin G; kb, kilobase(s).

' ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / Imprime an Canada

hand, the importance of positive charge in the N-terminal region has been demonstrated in prokaryotes (Inouye et al. 1982; Iino et al. 1987; Bosh et al. 1989; Sasaki et al. 1990). However, it has been suggested that this region is not impor- tant for the function of signal sequence in yeast invertase signal peptide (Brown et al. 1984; Kaiser and Botstein 1986). Contrary to this, Perlman et al. (1986) have demonstrated that the introduction of a positively charged residue in the N-terminal region of yeast invertase signal peptide resulted in the enhancement of the synthesis and secretion of invertase.

We have previously established the secretory system of HLY in yeast (Saccharomyces cerevisiae) using a synthetic HLY gene with a chicken lysozyme signal peptide gene (Jigami et al. 1986a). By using this system, the correlation between the processing efficiency and the (- 3, - 1)-rules (von Heijne 1985, 1986) was demonstrated (Nagahora et al. 1988), and the shift of cleavage site by the introduction of the turn-promoting amino acid Pro in the C-terminal region was indicated (Nagahora et al. 1988). Furthermore, we have investigated the molecular basis of the (- 3, - 1)-rules, show- ing that bulky residues at the - 1 position inhibit the

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DNA Polymerase I - (Klenow) - -

I S1 Nuclease F i l l i n I

1 ClaI 1 inker mlinkerz1 I=

I Gnpppd duplex I I i 7- I

pFJ-501 I

PFJ-502 P F J ! ~ O ~ I

pYS-201 I

pYS-202 ( L Y s ( - ~ ~ ) ) (Phe(-17)) (Glu(-17)) (Pro(-21, Ala(-1) (Pro(-2)) Val(-1))

FIG. 1. Strategy for the construction of chicken lysozyme signal peptide alteration and yeast expression plasmids for secretion of HLY.

cleavage of signal sequence by steric hindrance (Tsuchiya et al. 1993). In the present study, we have prepared 10 chicken lysozyme signal peptide variants that contained mutations in the N-terminal and the C-terminal regions, and elucidated the effect of the mutations o n the cleavage site, secretion of the HLY, and the efficiency of signal process- ing. Based upon the results of our current studies, we describe the structural requirements of the N- and the C-terminal regions of the signal sequence.

Materials and methods Strains

The Escherichia coli strain C600 (F - , thi-I, thr-I, leuB6, IacYI, tonA21, supE44) was used for bacterial transformation. Saccharo- myces cerevisiae strain kk-4 (MATa, ura3, his1 or his3, trpl, leu2, ga180) was used as the host strain.

Media The E. coli cells were grown in LB broth (1% Bacto-Tryptone,

0.5% yeast extract, 0.5% NaCI, 0.2% glucose). Ampicillin was added, where appropriate, to a final concentration of 50 pg/mL. The S. cerevisiae cells were grown in YPD medium (2% Bacto- Peptone, 1% yeast extract, 2070 glucose). Transformation of E. coli and S. cerevisiae was done by the methods of Cohen et al. (1972) and Ito et al. (1983). respectively. In the experiments to express the HLY gene, a synthetic medium was used containing 0.67% Bacto-Yeast nitrogen base without amino acid, supplemented with an amino acid mixture (20-375 pg/mL) lacking leucine.

Enzymes and chemicals Restriction enzymes and T, polynucleotide kinase were pur-

chased from Toyobo (Osaka, Japan), T, DNA ligase was from Takara-Shuzo (Kyoto, Japan), and zymolyase lOOT was from Seikagaku-Kogyo (Tokyo, Japan). Micrococcus lysodeikticus was from Sigma (St. Louis, Mo.). Anti-HLY IgG (rabbit serum) was from Alpha Therapeutic Corporation (Los Angeles, Calif.). The other chemicals used were of analytical or biochemical reagent grade. Standard techniques of restriction endonuclease digestion, ligation, and gel electrophoresis were done as described by Maniatis et al. (1982).

DNAs Figure 1 shows the strategy for the construction of chicken lyso-

zyme signal peptide alterations and yeast expression plasmid for secretion of HLY. At first, pFJ-001 was constructed from HindIII/EcoRI fragment (5.5 kb) of the S. cerevisiae - E. coli shuttle vector YEp5l (Broach et al. 1983), the EcoRI-SalI (1 kb) and XhoI-BamHI (1.8 kb, carrying the ENOl promoter) fragments of pMC1587-ENO-A (Jigami et al. 1986b), and the BamHI- HindIII fragment (0.8 kb, carrying the chicken lysozyme signal peptide sequence and HLY structural gene) of pHLYSIG (Jigami et al. 1986a) joining by T, DNA ligase. The pFJ-001 has an addi- tional sequence coding 11 amino acid residues at the N-terminal of the chicken lysozyme signal peptide sequence. To mutate this additional sequence, this plasmid was cleaved by BamHI and Sun, and pFJ-002 was prepared by "fill in" using the Klenow fragment of DNA polymerase I. Several DNA linkers were inserted after treatment of S1 nuclease as follows: pFJ-003 by ClaI linker, pFJ-004 by Sac1 linker, pFJ-005 by HindIII linker. On the other hand, to remove this additional sequence, pFJ-001 was lysed by BamHI and Ba131, and pFJ-1053 was prepared. The SalI/HindIII fragments (0.8 kb) from pHLYSIG were mutated by gapped duplex method (Kramer and Fritz 1987) using synthetic DNA oligomer, and pFJ-501 and pFJ-503 were prepared by inserting them to pFJ-1053. The pFJ-502 was prepared by cassette replacement of SalI/TaqI fragment of pHLYSIG using synthetic double-stranded DNA following insertion of this fragment into pFJ-1053. The SalI/HindIII fragments (0.8 kb) from pFJ-1053 were mutated by the method of Kunkel(1985) and then pFJ-201 and pFJ-202 were prepared by the insertion into pFJ-1053. The sequence of the mutant gene was checked by dideoxy sequencing (Sanger et al. 1977). These multicopy plasmids containing the HL Y transcrip- tional units, which consists of yeast ENOl promoter (Uemura et al. 1986), chemically synthesized chicken lysozyme signal peptide gene with HL Y and a gene, 2-pm FLP terminator, were used to secrete HLY in yeast.

Measurement of HL Y secretion Yeast cells carrying the plasmid containing the HL Y transcrip-

tional units were cultivated in a flask for 4 days at 30°C on a reciprocal shaker, and the HLY activity was measured photometri- cally by a modification of the method of Morsky (1983). The assay sample (200 pL) was added to 800 pL of a cell suspension of

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TABLE 1. Amino acid sequence of each mutant signal and effect of mutations on the activity of HLY secreted from yeast cells

HLY activity Plasmid Structure of signal peptide (U/mL)

Chicken lysozyme signal- -Human lysozyme -17 -2-1 +1 +I30

pFJ1053 (WT) M R SLLILVLCFIPLAA L G KVFER....-.V 63.9

pFJ 001 (ExllDE) MALDPGEGVDSM R SLLILVLCFIPLAA L G KVFER--.--.V 0.0 pFJ 002 (Ex7D) MALDVDSM R SLLILVLCFIPLAA L G KVFER.---..V 5.8 pFJ 003 (Ex7) MALSMMSM R SLLILVLCFIPLAA L G KVFER--....V 51.1 pFJ 004 (Ex7R) MALRARSM R SLLILVLCFIPLAA L G KVFER....-.V 60.7 pFJ 005 (Ex6) MASSLSM R SLLILVLCFIPLAA L G KVFER-..--.V 58.1 pFJ 501 (LYs(- 17)) M K SLLILVLCFIPLAA L G KVFER......V 56.9 pFJ 502 (Phe(- 17)) M E SLLILVLCFIPLAA L G KVFER...-..V 14.1 pFJ 503 (Glu( - 17)) M E SLLILVLCFIPLAA L G KVFER.-....V 12.1

pYS 201 (Pro(- 2), Ala( - 1)) M R SLLILVLCFIPLAA P KVFER-.-..-V 33.0 pYS 202 (Pro(- 2), Val(- 1)) M R SLLILVLCFIPLAA P KVFER......V 14.6

NOTE: The amino acids are shown in capital letters and the numbering refers to the amino acid sequence of the chicken lysozyme signal peptide and human lysozyme. The amino acids that were substituted are underlined. The lysis of M. lysodeikticus cells was measured by adding supernatant of each culture. One unit of HLY is defined as the amount of enzyme that decreases 0.001 unit per minute at 25°C. WT, wild type.

M. lysodeikticus (0.15 mg/mL) in 50 mM potassium phosphate buffer (pH 6.5). The initial decrease in the absorbance at 450 nm of the mixture caused by the lysis of bacterial cells was measured at 30°C for 5 min. One unit (decrease of 0.001 in absorbance at 450 nm per minute) corresponds to 0.02 pg HLY/mL culture medium under our assay conditions.

N-terminal sequence Production and purification of HLYs was performed as

described by Jigami et al. (1986a). Secreted HLYs obtained from 500-mL cultures were purified in this manner and applied to a peptide sequencer (model 6600, MilliGen/Biosearch). The amino acid sequences were analyzed by measuring the amounts of phenyl- thiohydantoin amino acids to determine the cleavage sites of mutant signals.

Western blot analysis and processing of mutant preHLY Yeast cells harboring each mutant plasmid were cultured in a

synthetic medium containing 2% glucose. The cell extracts from the mid-log phase culture were loaded onto 15% polyacrylamide slab gels and electrophoresed. After electrophoretic transfer to nitrocellulose filters, antibody binding was carried out in a blocking buffer (1 Yo gelatin, 20 mM Tris, 500 mM NaCI, 0.05% Tween-20, pH 7.5) with 50 pL of HLY antiserum. Filters were washed with blocking buffer and filter-bound antibodies were labeled by incu- bation in blocking buffer containing 2 pCi of '25~-labeled protein A. Filters were washed and exposed to X-ray film at room tem- perature or - 80°C.

Processing efficiency of mutant preHLY was determined by den- sitometric scanning of the autoradiogram and expressed as a percent of mature HLY/(mature HLY + preHLY).

Results The amino acid sequence of the mutant signals prepared

in this study and their effects on HLY secretion are sum- marized in Table 1. To elucidate the importance of the posi- tive charge of the N-terminal region in the eukaryotic signal, we replaced Arg at position - 17 in the chicken lysozyme signal peptide with basic Lys (Lys(- 17)), hydrophobic Phe (Phe( - 17)), or acidic Glu (Glu(- 17)). The lytic activities of secreted HLYs in the mutants Phe(- 17) and Glu(- 17) were less than that of the wild type, in contrast to mutant Lys(- 17) which was similar to that of the wild type. Moreover, these results were related with the processing effi-

TABLE 2. Effect of mutation on processing efficiencies

Mutant Processing efficiency ('-70)'

Wild type ExllDE Ex7D Ex7 Ex7R Ex6 Lys(- 17) Phe(- 17) Glu(- 17)

T h e efficiency of signal peptidase to cleave preHLY was determined by densitometric scanning of the autoradiogram and expressed as a percent of mature HLY/(mature HLY + preHLY) as measured by Western blotting.

ciencies (see Table 2). The processing efficiencies of Phe(- 17) and Glu(- 17) were lower than those of wild type and Lys(- 17). These results indicate that the positive charge of this region plays an important role in the function of the eukaryotic signal, as well as the prokaryotic signal. On the other hand, the lytic activity level and processing efficiency did not seem to depend upon the amino acid species donating the charge in the N-terminal region, because the wild type (Arg(- 17)) and the mutant Lys(- 17) had nearly the same results of HLY activities and processing efficiencies.

To study the structural requirement of the N-terminal region, we elongated this region with five different additional sequences. Additional sequences containing three (Ex1 lDE) or two acidic residues (Ex7D) decreased the lytic activity of secreted HLY and processing efficiency (as did mutant Glu( - 17), but additional sequences containing only neutral residues (Ex6, Ex7) had little effect (see Table 1). This result shows that the length of this region scarcely influenced the function of the signal peptide. Additional positive charge in the elongated sequence (Ex7R, as well as the mutants Ex6 and Ex7) barely affected the activity of secreted HLY and processing efficiency (see also Table 1).

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TABLE 3. The signal cleavage sites in various preproteins

Mutant Structure of signal peptide and cleavage site

Chicken lysozyme signal- -Human lysozyme -18 - 6 -2 -1 + 1 +I30

Wild typea MRSLLILVLCFI P L A A L G K V F E R......V 100% t

pro( - 2) MRSLLILVLCFI P L A A P G K V F E R......V 880701 t 12%

Pro(-2),Ala(-1) MRSLLILVLCFI P L A A P A K V F E R......V 100% t

Pro(-2),Val(-1) MRSLLILVLCFI P L A A P K V F E R.-....V 100% I

NOTE: The amino acids are shown in capital letters and the pa sites and the ratios of each cleavage are shown by arrows and

'Previously prepared by Jigami et al. (19860). b~reviously prepared by Nagahora et al. (1988).

On the other hand, we attempted to control the cleavage site by the introduction of the turn-promoting Pro in the C-terminal region. Nearby cleavage sites, Leu at position -2 and Gly at position - 1, were replaced with Pro and Ala (Pro( - 2), Ala( - 1)) or Pro and Val (Pro( - 2), Val( - 1)) respectively. These mutations decreased the lytic activities of secreted HLYs (see Table I), but resulted in the cleavage of these signal peptides after Pro at position - 2 (see Table 3), though mutant Pro( - 2) had heterologous cleavage sites. We have thus succeeded in controlling the cleavage site by the introduction of a turn-promoting residue in the C-terminal region.

Discussion Recently, it has been reported that the positively charged

N-terminal region of the preprolactin signal sequence is not necessary for translocation (Andrews et al. 1992). However this may not apply to every eukaryotic signal sequence. In fact, Perlman et al. (1986) have shown that the introduction of positively charged amino acid residues to the N-terminal region of yeast invertase signal peptide enhances the activities of both intracellular and extracellular invertase. In our study, removal of positive charge (Phe(- 17)) or introduc- tion of negative charge (ExllDE, Ex7D, Glu(- 17)) in the N-terminal region resulted in the decrease or loss of the lytic activity of secreted HLY and a decrease in processing effi- ciency. The lytic activity of HLY and processing efficiency were positively correlated. As the lytic activity of HLY reflects the amount of HLY secreted from yeast cells, we believe that these mutations influence the secretion of HLY by inhibition of signal peptide processing. In prokaryotes, it has been suggested that the positively charged domain of the signal peptide is required for the binding of precursor proteins via SecA to the membrane surface to initiate trans- location (Sasaki et al. 1990). In addition, Killian et al. (1990) have shown that the negative charge in cell membrane lipids is essential for a functional interaction of signal peptide with the membranes (Killian et al. 1990). Taking these reports and our results into consideration, we suggest that the posi- tively charged N-terminal region is involved in the interaction with the negatively charged surface of endoplasmic reticulum at the first step of translocation in eukaryotes as well as prokaryotes. That is, the positive charge in the N-terminal region promotes the penetration of preprotein into the mem- brane of the endoplasmic reticulum by ionic interaction

~sitions of the replaced amino acids are underlined. The cleavage percentages, respectively.

between its positive charge and the negative charge on the surface of the membrane.

On the other hand, considering the result of Ex7R, the number of residues in the N-terminal region has little influence on the function of the signal peptide. Only mini- mum positive charge (a single basic amino acid residue) seems to be needed in this region of chicken lysozyrne signal peptide. Compared with prokaryotic signal peptides, the positive charge in the N-terminal region appears to be less important in eukaryotes. It has been suggested that eukaryotic signal peptides are initiated by an unformylated, positively charged Met residue, which can supply the positive charge instead of Lys or Arg in the N-terminal region (von Heijne 1984). Many eukaryotic signal peptides certainly have no positively charged residue in this region, but it is thought that they can function by using the positive charge of N-terminal Met. In addition, Hikita and Mizushima (1992) have shown that the requirement of positive charges in this region can be compensated by a longer hydrophobic region of the signal peptide in prokaryotes.

As shown in Tables 1 and 2, the change of length of the N-terminal region did not significally affect the lytic activity of secreted HLY or the processing efficiency, except for the negative charged mutants. Generally, the length of eukaryotic N-terminal region is short. In most cases, it composed of only two residues (Met-Lys- or Met-Arg-) (von Heijne and Abrahmskn 1989), which suggests that the role of this region is to supply a positive charge. Extra residues may have little influence on the function of signal peptide unless they inhibit the interaction between Lys and the membrane of the endoplasmic reticulum.

The mechanism of signal cleavage has not yet been fully elucidated in eukaryotes. However, it has been proposed that the 0-turn structure around the cleavage site is essential for the processing of the signal sequence in the E. coli prolipoprotein (Inouye et al. 1986), although others have shown that a potential p-turn structure in the C-terminal region is not required for processing (Laforet and Kendall 1991). We have previously shown a shift of the cleavage site by replacing Leu at position - 2 with Pro in the chicken lyso- zyme signal peptide (Pro(- 2), see Table 2) (Nagahora et al. 1988). This resulted in the heterologous cleavage sites as follows: 88% was after Pro at position -2 and 12% was after Gly at position - 1. We hypothesize that this is due to Gly at position - 1, because Gly is both a turn promoter

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as well as Pro. If this is correct, the replacement of Gly at position - 1 with another amino acid residue, which is not turn promoting, is expected to result in a complete cleavage after Pro at position - 2. As shown in Table 3, we succeeded in the complete shift of cleavage site by the replacement of Gly at position - 1 with nonturn-promoting Ala or Val. This demonstrates that the cleavage site can be controlled by the introduction of a turn-promoting residue like Pro.

Acknowledgements This work was supported in part by a grant from the

Ministry of Agriculture, Forestry and Fisheries. We thank Dr. C.R. Youngs for critically reading the manuscript.

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