Cell, Vol. 66, 541-554,

August

9, 1991, Copyright

0 1991 by Cell Press

A Novel Endopeptidase from Xenopus That Recognizes a-Helical Secondary Structure Nicole M. Resnick,’ W. Lee Maloy,t H. Robert Guy,* and Michael Zasloff ‘The Division of Human Genetics Children’s Hospital of Philadelphia and the Molecular Biology Graduate Group University of Pennsylvania Philadelphia, Pennsylvania 19104 tMagainin Sciences, Inc. 5110 Campus Drive Plymouth Meeting, Pennsylvania 19462 *Laboratory of Mathematical Biology National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 l

Summary The magainln peptides of Xenopus laevis are broadspectrum antimicrobial agents. Upon discharge from the skin glands, these basic, amphipathfc peptldes are each further processed at a single Xaa-Lys bond into half-peptides by a cosecreted protease. We describe the characterization and purification to homogeneity of this endopeptidase from Xenopus skin. The enzyme is a metalloprotease 110 kd in size. Analyses of substrate specificity revealed that the endopeptidase recognizes peptldes that share the ability to adopt an amphipathic, a-helical motif composed of at least 12 residues, with one face strongly hydrophobic. Cleavage occurs on the amino side of a specific lysine that must be precisely positioned relative to the hydrophobic face of the a helix. This enzyme, which we propose to call ‘magaininase,” represents a novel class of endopeptidases that hydrolyzes peptides on the basis of specific secondary structure rather than primary amino acid sequence. lntroductlon The magainin family of antibiotic peptides, first isolated from the skin of Xenopus laevis (Zasloff, 1987) comprises at least a dozen basic, ionophoric peptides, including magainins 1 and 2, PGLa, xenopsin precursor fragment (XPF), and caerulein precursor fragment (CPF) (for reviews see Bevins and Zasloff, 1990; Boman, 1991). The peptides are produced in the granular glands, specialized secretory cells that store large amounts of biologically active peptides and neurotransmitters. The granular glands are present in amphibian skin (Dockray and Hopkins, 1975) and stomach (Moore et al., submitted) and release their contents in a holocrine fashion upon stress or injury. These glands are believed to serve physiological roles in defense against macroscopic predators and in microbial control following wounding. Strikingly, most hormones and many of the processing enzymes involved in their biosynthesis, stored in the anuran granular gland, have

been found in the central and diffuse peripheral nervous systems of mammals (Bevins and Zasloff, 1990). Isolation of magainin cDNAs from Xenopus skin indicated that, similarto mammalian hormonesand neuropeptides, the magainin peptides are initially synthesized as large polyproteins. In the case of amphibian skin polyproteins, proteolytic events serve to liberate both antibiotic and hormonally active peptides (Sures and Crippa, 1984; Richter et al., 1986; Poulter et al, 1988). The peptides contained within the granular gland are stored in secretory vesicles as processed active species, suggesting that initial proteolytic events occur prior to secretion (Gibson et al., 1986). However, mass spectroscopic analysis of Xenopus gland secretions revealed that following discharge from the granular glands, the peptides undergo further proteolysis, resulting in half-peptide fragments that no longer exhibit antibiotic activity (Gibson et al., 1986; Giovannini et al., 1987). To date, several of the amphibian skin proteases believed to be responsible for the processing of the magainin propeptides have been isolated. Synthetic peptides containing the putative recognition sites for processing enzymes have been utilized as substrates to purify sequence-specific proteases from Xenopus (Mizuno et al., 1986; Mollay et al., 1986; Kuks et al., 1989; Darby et al., 1991). Characterization of these enzymes has revealed additional similarities between mammalian and amphibian processing systems. Examination of endoproteolytic activities in particular has demonstrated that recognition sequences bracketing the biologically active peptides, such as dibasic and monobasic residues, have been conserved across species (Schwartz, 1986; Darby and Smyth, 1990). One intriguing aspect of the sequence-dependent endopeptidases believed to recognize amino acid motifs is their ability to hydrolyze selected peptide bonds while leaving other consensus sites uncleaved. Furthermore, reports of tissue-specific processing demonstrate that endoproteases may differentially cleave a particular peptide bond in various tissues (Pate1 et al., 1981; Zakarian and Smyth, 1982). Clearly, additional parameters determine substrate recognition by endopeptidases, and it is believed that higher order structure must play a critical role (Plevrakis et al., 1989; Docherty et al., 1989; Brakch et al., 1989). While investigating the processing system utilized by Xenopus laevis in the generation of antimicrobial peptides, we came to focus our attention on the activity responsible for the in vivo hydrolysis of the magainin family of peptides. In this report we describe the purification and characterization of the enzyme that cleaves the magainins into half-peptides. The enzyme responsible for the precise endoproteolytic processing of these basic, amphipathic molecules is a 110 kd metalloprotease. After establishing that the purified enzyme cleaves several natural peptides of the magainin family despite their widely different primary sequences, we decided to investigate the parameters governing substrate specificity. Through the utilization of synthetic substrate analogs, we found that the enzyme

Cdl 542

A.

P(aturdsubstratesfLQm

Xenooum

c.

&g&&&aomissionanaloeues (Des-l to Des-23)

4 magainin

2

GIGKFLESAK

Relative

KFGKAFVGEIbfNS

4 PGLa

GWASKAGAIAG

XPF

GWASKIGQTLG

CPF

GFASFLGKAL

KIAKVALKAL 4

KIAKVGLKELIQPK

4

B.

Magainin

KAALKIGANLLGGTPQQ

2 -1

truncations* Relative

Des-l

-1GKFLHSA~GKAFVGEIMNS

Des-l-2

--GKFLHSAKKFGKAF~GEIMNS

Des-l-3

---KFLHSAKKFGKAFVGEIMNS

Des-l-4

----FLHSAKKFGKAFVGEIMNS

cleavage +

D.

Des-22-23

GIGKFLHSA~FGKAFVGEIM--

+++

Des-20-23

GIGKFLHSAuFGKAFVGE----

+++

Des-18-23

GIGKFLESAuFGKAFV------

+++

Magalnrn

. .

2 amino-terminal

extension.* 4

REVR(l-4) E.

PGLa

REVRGIGKFLHSAKKFGKAFVGEIMNS pmissioq

Relative

F.

G.

GMAS

Des-Glyll

GMASKAGAIA

Des-Lys12

GMASKAGAIAG

AGAIUIAKVALKAL

cleavage ++++ +t

KIAKVALKAL IAKVALKAL

Maeaininm&substitutions KllR

GIGBFLBSA@$GBAFVBZ&NS

+t

KllE

GIGKFLHSAGBFGKAFVGEIMKS

-

KllA

GIGKFLHSAGPFGKAFVGEIMKS

-

KllP

GIGKFLHSAGBFGKAFVGEIMKS

-

GlOA

GIGKFLHSAP&FGKAFVGEIMKS

+++

GlOE

GIGKFLHS~FGKAFVGEIMKS

++++

GlOP

GIGKFLBSAE'KFGKAFVGEIMKS

-

Miscellaneous-M melittin

.

.

GIGAVLKVLTTGLPALISWIKKKKQQ

beainin

cleavage

+ + + f + + + + + + + + ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

~gJutama&&utitution~

BIGKFLHSAGKFGKAFVGEIMKS

-

GBGKFLHSAWGKAFVGEIMKS GIBKFLHSAWFGKAFVGEIMKS GIGBFLHSAmFGKAFVGEIMKS GIGKBLHSA-GKAFVGEIMKS GIGKF@tHSAmGKAFVGEIMKS GIGKFLBSAEgFGKAFVGEIMKS GIGKFLHBAWGKAFVGEIMKS GIGKFLESBG&FGKAFVGEIMKS GIGKFLHSmFGKAFVGEIMKS GIGKFLBSAGEFGKAFVGEIMKS GIGKFLHSAGKBGKAFVGEIMKS

++ t+ +++ +

+++t ++++ +++ + +++ -

+++

analagues

Des-LysS

IGKFLHSUFGICAFVGEIMNS G GKFLHSAggFGKAFVGEIMNS GI KFLHS~FGKAFVGEIMNS GIG FLHS~FGKAFVGEIMNS GIGK LHSAgICPGKAFVGEIMNS GIGKF HSA&&FGKAFVGEIMNS GIGKFL SAggFGKAFVGEIMNS GIGKFLE ALIHFGKAFVGEIMNS GIGKE'LES =FGKAFVGEIMNS GIGKFLHSA KFGKAFVGEIMNS GIGKFLHSm FGKAFVGEIMNS GIGKFLHSAU GKAFVGEIMNS GIGKFLHSAI(pF KAFVGEIMNS GIGKFLHSAUFG AFVGEIMNS GIGKFLHSAmFGK FVGEIMNS GIGKFLHSAmFGKA VGEIMNS GIGKFLHSAnFGKAF GEIMNS GIGKFLESAmFGKAFV EIMNS GIGKFLHSABgFGKAFVG IMNS GIGKFLHSAgELFGKAFVGE MNS GIGKFLHSAKKFGKAFVGEI NS GIGKFLHSAUFGKAFVGEIM S GIGKFLHSAmFGKAFVGEIMN

oeDtides -

Figure Tested

1. Summary of Peptides against Endopeptidase

and Analogs

Arrows denote cleavage sites identified by reverse-phase HPLC and amino acid analyses ([A] and [B]) as described in Experimental Procedures. Underlined residues denote cleavage sites identified by acid PAGE. Amino acids deleted from peptide termini are represented as dashes (B), while residues internally omitted are denoted by a space in the full-length sequence ([Cl and [El). Substituted amino acids are represented as highlighted, reversed-type single letters ([O] and [F]). Analogs labeled as “Des-x” signify that residue x is deleted. The column on the right summarizes the relative cleavage of each peptide analog relative to endopeptidase activity against native magainin P-amide. Four plus signs indicate wild-type levels of cleavage, while a minus sign indicates no cleavage. All determinations of relative cleavage were made by visualizing reaction products separated by acid gel electrophoresis and stained in Coomassie blue as described in Experimental Procedures. All peptides listed are synthesized as terminal carboxy-amidated molecules, with the exception of those denoted by an asterisk.

Endoproteolytic 543

Cleavage

of Amphipathic,

a-Helical

Peptides

(Enzvme

cleaves peptides capable of forming a particular structural motif. The novel endopeptidase described in this report is demonstrated to hydrolyze peptides on the basis of their ability to adopt an amphipathic, a-helical conformation. We propose to call this novel enzyme “magaininase” and anticipate that it will teach us about the requirements of other highly selective proteases for which the structural determinants directing substrate specificity are presently unknown.

Standards

units) t I

10

5

1

0.5

0.1 d.

12

3

4

,’

5

6

7

-

Magainin

2(11-23)

-

Magainin

2(1-23)

-

Magainin

2(1-10)

0

Results Figure

Previous analyses of the magainin peptides isolated from the skin secretions of Xenopus laevis revealed that following discharge of the granular glands, the mature bioactive peptides are further processed. Fast atom bombardment mass spectroscopy identified the precise site of cleavage of each peptide, denoted by arrows in Figure 1A (Giovannini et al., 1987). To understand the regulation and metabolic fate of the magainins, we decided to isolate the endopeptidase(s) responsible for generating the half-peptide products. Purification of the Endopeptidase Beginning with Xenopus laevis skin extract, theendopeptidase was purified to homogeneity by several chromatographic steps. Enzymatic activity was monitored by acid PAGE (Gabriel, 1971; Boman et al., 1989) of the reaction products generated by cleavage of magainin 2, as illustrated in Figure 2. Differences in net electrostatic charge result in the resolution of full-length magainin 2 peptide substrate from the two cleavage products (amino-terminal half-peptide, magainin l-10, and carboxy-terminal halfpeptide, magainin 1 l-23). The gel displays increased production of half-peptides, with a concomitant decrease in the full-length substrate as additional units of enzyme activity are added to a reaction (Figure 2, lanes l-5). Both cleavage products comigrate with the synthetic half-peptides run as standards on the gel (Figure 2, lanes 6-8). A summary of the purification reveals that the endopeptidase was purified approximately 1 OO-fold starting from the ammonium sulfate fractionation (Table l), reflecting the abundance of the enzyme in Xenopus skin. Figure 3 is a silver-stained sodium dodecyl sulfate (SDS)-polyacryl-

Table

1. Summary

of Endopeptidase Volume (ml)

Purification

Step

Ammonium

Sulfate

3.6

Isoelectric Focusing Sephacryl S-300 Hydroxyapatite Glycerol Gradient

15.5 10.6 4.2 1.6

2. Enzymatic

Activity

as Monitored

by Acid PAGE

Endoproteolytic activity was monitored at each stage of purification by acid PAGE. Shown is the resolution of magainin 2 substrate and half-peptide cleavage products on a 15% acrylamide acid gel prepared and run at pH 4. The gel was then stained with Coomassie blue R-250 to visualize the peptides as described in Experimental Procedures. Lane 6 demonstrates the migration of synthetic full-length magainin 2 peptide (l-23). lane 7 shows the synthetic amino-terminal half-peptide (l-lo), and lane 6 shows the synthetic carboxy-terminal half-peptide (1 l-23). The products of cleavage reactions utilizing 50 pg of magainin 2 substrate and decreasing amounts of enzyme units are shown in lanes l-5. One unit of enzyme activity (lane 3) is defined as described in Experimental Procedures.

amide gel illustrating each purification final homogeneous species.

step leading to the

Characterization of the Endopeptidase The endopeptidase was found to have a molecular weight of approximately 110,000. When separated by SDS-polyacrylamide gel electrophoreseis (PAGE) under both reducing and nonreducing conditions, the protein band corresponding to enzymatic activity throughout every stage of purification migrated to the same position, suggesting that the activity is composed of a single subunit. Sedimentation of enzymatic activity in a glycerol gradient subjected to rate zonal centrifugation indicated that it has a molecular mass of about 110 kd and provided additional evidence that a monomeric species is responsible for endoproteolysis (data not shown). Inhibition studies employing several proteinase inhibitors suggested that the endopeptidase is a metalloproabolished tease (Table 2). EDTA and 1 ,lO-phenanthroline enzyme activity. Endoproteolysis was restored by the sub-

Purification Protein P-w/ml)

(mg)

U/ml’

Total Units

15

54

20,000

72,000

1,333

2,250 3,000 4,000 2,000

34,675 31.800 16,800 3,200

15,163 43,000 134,400 133,333b

0.15 0.07 0.03 0.015

Total Protein

2.3 0.74 0.126 0.024

Specific

Activity

All values represent the average of five separate purifications. B One unit of enzyme activity as described in Experimental Procedures. 1 The decrease in specific activity from the hydroxyapatite step to the glycerol gradient step is a result of quantitating sample (only a subset of the active fractions recovered from the glycerol gradient contained pure enzyme).

Yield (%)

Fold

100

-

48 44 23 4.4

11.4 32.3 100.8 100

only homogeneous

enzyme

Cell 544

M

AS

IEF

SL

HPT

GG -

123456 200

-

200 116 97.4 66

11697.4 66 -

Figure cation

3. SDS-PAGE

of Enzyme

Material

at Each

Stage

25 26

sequent addition of any of several metals including zinc, magnesium, cobalt, and manganese (data not shown). In contrast, serine protease inhibitors such as leupeptin, phenylmethylsulfonyl fluoride, and TPCK, cysteine protease inhibitor E-64, and the aspartyl protease inhibitor pepstatin had no effect on enzyme activity. Endoproteolysis was insensitive to bacitracin and phosphoramidon as well. Initial kinetic analyses utilizing magainin 2-amide substrate at various concentrations revealed that under standard assay conditions, the endopeptidase exhibits a kcatof approximately 50 s-’ and a K, value for this substrate of less than 20 PM. The endopeptidase has an estimated isoelectric point of 5.3 and exhibits optimal activity over a pH range of 6-6 (data not shown).

2. Inhibition

Profile

-

PGLa

2

27

Figure 4. A Single strates

28

Enzyme

29

30

31

Is Active

32

33

against

Several

Peptide

Sub-

Active fractions from the hydroxyapatite column were pooled and ap plied to a 15%-300/o glycerol gradient as described in Experimental Procedures. Fractions from the glycerol gradient were assayed for endoproteolytic activity against several of the natural antimicrobial peptides. (A) Silver-stained SDS-PAGE of fractions 25-33 recovered from the glycerol gradient. The arrow along the top indicates the direction of sedimentation. The protein size markers (lane M) are in kilodaltons. Endoproteolytic cleavage of magainin P-amide(E), PGLa (C), and XPF (D) is detected by acid PAGE followed by Coomassie blue staining. The number denoting the glycerol gradient fraction at the bottom indicates the source of enzyme utilized for each individual assay. The arrows denote the migration of each full-length uncleaved peptide substrate, and the disappearance of that band in each gel indicates conversion to half-peptide products. In the case of both PGLa and XPF, cleavage by the endopeptidase generates an amino-terminal halfpeptide with no net electrostatic charge, thereby precluding its migration into the gel.

of Endopeptidase

Inhibitor

Concentration

Percent

Leupeptin Pepstatin PMSF TPCK E-64 Bacitracin Phosphoramidon

1 100 10 1 300 1

0 0 0 0 0 0 0 0 25 25 50 70 100 100

l,lO-Phenanthroline

Magainin

of Purifi-

Enzyme purity was assessed by SDS-PAGE of protein fractions from the various stages of purification. Lane 2, 15 ug (approximately 20 U) of the ammonium sulfate fraction; lane 3,1.5 pg (20 U) of the isoelectric focusing fraction; lane 4,1 .O ug (20 U) of the Sephacryl S-300 fraction; lane 5,0.5 ug (20 U) of the hydroxyapatite fraction; lane 6,0.2 ug (10 U) of the glycerol gradient fraction. The molecular weights (in kilodaltons) of marker proteins in lane 1 are indicated. Samples were separated by electrophoresis as described in Experimental Procedures and stained with silver nitrate. The two faint bands migrating at approximately 6065 kd in lane 6 are artifacts commonly detected by silver stain methodology (Ochs, 1963).

Table

-

mM t&l mM mM pM mglml

1 PM 100 pM 0.5 mM 5mM 50 mM 0.2 mM 2mM 20 mM

Inhibition

The percent inhibitions listed were determined by Coomassie blue staining of cleavage products utilizing magainin P-amide as substrate following acid gel electrophoresis. One unit of purified endopeptidase was incubated with inhibitor at the above concentrations for 1 hr and then incubated with native substrate under standard assay conditions.

We next sought to determine if the endopeptidase was responsible for cleaving other members of the magainin peptide family. Fractions recovered from a 150/o-30% glycerol gradient were assessed by SDS-PAGE (Figure 4A) and tested against magainin P-amide, PGLa, and XPF peptides (Figures 48,4C, and 4D). Acid gel electrophoresis revealed peak activity against magainin P-amide in fractions 27-30, which corresponded to the highly enriched protein band migrating at approximately 110 kd (Figure 4A). Maximal endoproteolytic activity against PGLa and XPF also correlated with the 110 kd protein of fractions 27-30, leading us to conclude that a single enzyme hydrolyzes several peptide substrates. Characterization of each peptide’s precise cleavage site demonstrated that the enzyme is an endopeptidase. Following incubation of 1 U of pure enzyme with a particular substrate, reactions were analyzed by reverse-phase high

Endoproteolytic 545

Figure

Cleavage

5. The Enzyme

of Amphipathic,

a-Helical

Peptides

Is an Endopeptidase

Standard cleavage reactions testing magainin Z-amide (A), PGLa (E), and XPF (C) as substrates against 1 U of purified enzyme were analyzed by reverse-phase HPLC. Peak a in each chromatograph was found by subsequent amino acid analyses (described in Experimental Procedures) to represent the amino-terminal half-peptide product, peak b represents the carboxy-terminal half-peptide product, and peak c represents the uncleaved full-length peptide substrate. Reversephase HPLC analysis was performed as described in Experimental Procedures.

pressure liquid chromatography (HPLC) (Figure 5). The major peptide peaks resolved by HPLC were then subjected to amino acid analysis in order to identify the sites of endoproteolysis (data not shown). The internal Lys-Lys peptide bond at residues 10 and 11 of magainin 2-amide was hydrolyzed by the enzyme, confirming the cleavage site originally reported in vivo (Giovannini et al., 1987). HPLC chromatographs of reactions utilizing PGLa and XPF as substrates also indicated that a single proteolytic event was directed against each peptide (Figures 58 and 5C). Amino acid analyses of each reverse-phase peak revealed that the endopeptidase cleaved the expected Gly 11 -Lys 12 peptide bond of PGLa and XPF, as well as the analogous Leu lo-Lys 11 bond present in CPF (data not shown). Figure 1A summarizes the cleavage sites identified for each natural peptide substrate. Primary Sequence Alone Cannot Account for Recognition by the Endopeptidase Our data suggest that a single proteolytic enzyme can hydrolyze a family of functionally related peptides, each at a single peptide bond. While magainin, PGLa, XPF, and CPF all exhibit antimicrobial activity, these peptides do not share any extensive amino acid sequence identity. Only 2

residues are conserved in all four natural peptides so far demonstrated to undergo cleavage by the endopeptidase: a glycine at the amino terminus and a lysine on the carboxy1 side of the hydrolyzed bond. We found it most intriguing that peptides of such varied primary sequence are all recognized by one endoprotease and decided to ask the question, “How is specificity achieved?” Based on the data thus far presented, several possible determinants of substrate specificity can be envisioned. The endopeptidase may be employing a counting mechanism, whereby it always cleaves the peptide bond located a particular number of residues from the terminus of a substrate, regardless of the nature of the bond at that position. Another hypothesis is that endopeptidyl activity is dictated solely by primary sequence. The majority of reports on endopeptidases attribute substrate specificity to the presence of a particular amino acid or sequence of residues. Dibasic recognition sites, for example, are quite prevalent in propeptide precursors that undergo multiple processing events, and a large body of evidence supports the theory that endopeptidases recognize and cleave ArgArg or Arg-Lys peptide bonds (Darby and Smyth, 1990; Loh, 1988; Gluschankof and Cohen, 1987). Finally, another possibility involves the influence of substrate secondary structure on recognition and hydrolysis by an endoproteolytic enzyme. To explore the hypothesis regarding a counting mechanism, synthetic magainin 2 analogs with truncations of either amino- or carboxy-terminal residues were tested as substrates for the enzyme (see Figure 1 B for peptide sequences). If the endopeptidase is strictly measuring a certain number of amino acids from an end, each analog should be hydrolyzed, yet the site of cleavage should be shifted. As illustrated in Figure 8, slight cleavage of the Des-l analog was detected (lane l), while analogs with amino-terminal truncations of any greater length resisted cleavage by the endopeptidase (lanes 2-4). Deletion analog Des-l-3 (lane 3) is the most powerful demonstration that the endopeptidase does not invoke measuring of

1

23456

Figure 6. Magainin 2 Amino-Terminal Endoproteolytic Attack

7

Truncation

Analogs

Resist

Standard enzyme assays were conducted to determine whether the endopeptidase can hydrolyze magainin 2 amino- and carboxy-terminal truncation analogs. Cleavage was monitored by acid gel electrophoresis and Coomassie blue staining. Lane 1 illustrates slight cleavage of analog Des-l, lanes 2-4 (Des-l-2 through Des-l-4 analogs) demonstrate the absence of any half-peptide products, while lanes 5-7 (Des22-23, Des-20-23, and Des-16-23, respectively) reveal the liberation of half-peptide products as denoted. The fastest moving half-peptide represents the amino-terminal magainin l-10 fragment, while the slower-moving half-peptide represents the carboxy-terminal fragment.

Cell 546

= C-term. half Full length -

N-term.

half

GIGKFLHSAKKFGKAFVGEIMNS Figure

7. Single Amino Acid Omissions

in the Amino-Terminal

Half of Magainin

2 Result

in Peptides

That Are Resistant

to Endopeptidase

Activity

The single letter amino acid code below each lane denotes the residue omitted from the synthetic magainin 2 analog tested for cleavage by the endopeptidase in that reaction. Reaction products were resolved by acid gel electrophoresis and visualized by Coomassie blue stain. Analogs susceptible to attack are revealed by the appearance of the amino-terminal and carboxy-terminal half-peptides as labeled on the right.

amino acids from an end. Deletion of the first 3 residues from the amino terminus of magainin 2 serves to translocate the Gly 13-Lys 14 bond to positions 10-l 1 of the shortened peptide, where the endopeptidase normally cleaves. Although Gly-Lys represents the natural cleavage sequence recognized by the endopeptidase in native PGLa and XPF, this magainin analog is not hydrolyzed. In contrast, the carboxy-terminal truncation analogs were all cleaved to an extent comparable to wild type, even when as many as 6 residues are deleted (Figure 6, lanes 5-7). The common peptide fragment visualized in lanes 5, 6, and 7 comigrates with the native magainin 2 aminoterminal half-peptide (magainin l-lo), indicating that endopeptidase faithfully recognizes the Lys lo-Lys 11 bond. Another informative analog that we tested against the purified endopeptidase was magainin 2 REVR(l-4), possessing a 4 residue amino-terminal extension (see Figure 16). This analog was cleaved with approximately 50% efficiency relative to wild-type magainin 2, and HPLC and amino acid analyses confirmed that hydrolysis occurs at the native Lys-Lys bond despite its repositioning at residues 14-15. From these assays utilizing shortened and elongated magainin analogs, we conclude both that the endopeptidase does not achieve its cleavage specificity by measuring from a peptide terminus and that the determinant responsible for conferring recognition resides within the amino-terminal portion of the magainin peptide. To investigate which, if any, specific residues are critical for recognition and cleavage by the endopeptidase, a complete series of synthetic magainin 2 single residue omission analogs was assayed (Figure 7). This experiment demonstrates in dramatic fashion that susceptibility to hydrolysis cannot be attributed to any one particular amino acid. Omission of any single amino acid from positions 1 (Gly 1) through 12 (Phe 12) of the magainin 2 sequence resulted in equally decreased levels of endoproteolysis. On the other hand, analogs in which a single residue is omitted from position 14 (Lys 14) through 23 (Ser 23) were hydrolyzed at wild-type levels. The omission of Gly 13 appeared to confer an intermediate effect, which is perhaps explained by its position at the boundary of the domain recognized by the endopeptidase for cleavage. Figure 1C lists each peptide sequence and establishes that resistance to hydrolysis may be attributed to the omission of residues as diverse as glycine, lysine, and phenylalanine. The sole unifying characteristic is that each amino acid capable of rendering a substrate inactive through its omis-

sion is located within the amino-terminal half of the peptide. In addition to ruling out the hypothesis that primary sequence is the sole parameter governing substrate specificity, these data further support our belief that magainin’s recognition determinant resides within the first 12 residues of the peptide.

The Endopeptidase Recognizes Substrate Secondary Structure Despite their negligible primary amino acid sequence identity, the magainin peptides have been demonstrated to share structural features. Might these conformational similarities play a role in substrate specificity? Raman, nuclear magnetic resonance, and circular dichroism spectroscopy have revealed that in a phospholipid bilayer, the peptides adopt amphipathic, a-helical conformations (Matsuzaki et al., 1969; Williams et al., 1990; Duclohier et al., 1969; Bechinger et al., 1991). The potential a-helical nature of the amino-terminal portion of each substrate tested against the endopeptidase can be appreciated by examining molecular models of these sequences (Figure 8). The continuous registry of hydrophobic residues constituting the nonpolar face of each amphipathic helix (located along the right side of each view) represents a recurrent motif. The position of the endopeptidase cleavage site (denoted in pink) on the hydrophilic side signifies another shared feature of the natural substrates. Finally, the lysine residue located on the carboxyl side of the hydrolyzed bond (denoted by an arrow) projects from each helix in a similar orientation. The results of the magainin 2 omission analogs (Figure 7) can be explained when interpreted within this structural context. The omission of a single residue might not detract from the overall amphipathic nature of an a-helical configuration; however, the deletion of any amino acid within the first 12 would perturb the registry of hydrophobic residues along the nonpolar face, as well as their orientation with respect to the cleavage site. To challenge the hypothesis that the endopeptidase interacts with its substrates as modelled in Figure 8, a series of 12 synthetic magainin 1 substitution analogs, each representing the replacement of amino acids l-l 2 individually by a glutamic acid (Figure 1D), was assayed for endoproteolysis (Figure 9A). Substitutions of those residues believed to reside along the hydrophobic face were predicted to render an analog inactive, while substitutions

Endoproteolytic 547

Figure

Cleavage

6. The Natural

of Amphipathic,

Substrates

Modelled

a-Helical

Peptides

as a-Helices

Three-dimensional space-filling models of each natural peptide substrate reveals structural similarities despite their variable amino acid sequences. The top panel shows projections along the helical axis of re$dues 1-12 of magainin 2 and CPF and residues 1-13 of PGLa and XPF (residues constituting the carboxyl half of each peptide were not found to be critical for cleavage by the endopeptidase; see Figures 5 and 6). The bottom panel shows side views of the a-helical rods, whereby the amino terminus is at the very bottom and the carboxyl terminus is at the top of each half-peptide. The peptide backbone is colored white, and side chain carbons are colored green, sulfurs yellow, oxygens red, and nitrogens blue. The hydrolyzed bond in the peptide backbone is depicted in pink, and the lysine on the carboxyl side of each scissile bond is denoted by an arrow.

serving to maintain the hydrophilic face of a putative a-helix were not expected to profoundly affect substrate susceptibility. The results presented in Figure 9A suggest that hydrophobicity is a crucial determinant of substrate-enzyme interaction. More specifically, the peptide became a less favorable substrate upon the replacement of a glutamate for the 2 hydrophobic residues surrounding the Gly 10-Lys 11 cleavage site (Ala 9 and Phe 12; Figure 9A, lanes 9 and 12), as well as for the amino-terminal Gly 1 and Phe 5 (lanes 1 and 5; compare with lanes 6, 7, and 10 representing analogs cleaved at wild-type levels). As summarized in Figure 98, these particular residues all reside on the nonpolar face of the helix (blue amino acids) when projected on the axial and side view models of magainin 1-12. Conversely, with the exception of Lys 4 and Lys 11, substitutions of amino acids on the polar face of the helix (depicted in yellow) did not significantly inhibit hydrolysis of a substrate. If the endopeptidase is examining solely primary sequence at the cleavage site, substitutions of amino acids located at a distance from the hydrolyzed bond (Gly 1 and Phe 5) should not affect endopeptidase activity. Further-

more, if the enzyme requires only a certain peptide length relative to the cleavage site, unlike the magainin 2 omission analogs, glutamate substitutions should not hamper cleavage. What emerges instead is the apparent requirement of a particular substrate conformation. The quantitative differences in substrate susceptibility exhibited in this experiment serve to highlight those residues composing the recognition motif. While the 2 hydrophobic residues bracketing the cleavage site appear to be most crucial, 2 additional nonpolar amino acids (Gly 1 and Phe 5) must also be involved, because their replacement with a charged residue affects hydrolysis. What unifies these seemingly random amino acids is their potential to generate a strongly hydrophobic surface when the aminoterminal portion of the magainin peptide is configured as an a-helix. Three synthetic PGLa derivatives representing deletions of either Lys 5, Gly 11, or Lys 12 (see Figure 1E for sequences) were tested against the endopeptidase to further challenge our model of substrate secondary structure recognition. As demonstrated in Figure lOA, the DesLys 5 analog was cleaved as efficiently as native PGLa

Cell 546

Figure 9. Substrate Hydrophobicity minant of Recognition

3

Full length

peptide

-

N-termmal

half

GIGKFLHSAGKF

(Figure 1OA, lane 2) while the Des-Gly 11 PGLaderivative exhibited a decrease in endoproteolysis compared with native peptide (lane 4). The Des-Lys 12 analog was completely resistant to hydrolysis, as indicated by the equal intensity of uncleaved peptide in both the absence and presence of enzyme (compare lanes 5 and 6). Examination of helical wheel projections (Figure 1OB) of each derivative provides an explanation of why the DesLys 5 derivative remains an optimal substrate, but the Des-Gly 11 derivative exhibits compromised activity. Despite the deletion, the continuity of adjacent hydrophobic residues (designated by circles) in the Des-Lys 5 analog remains intact, as does their position directly across the helix from the cleavage site (compare with native PGLa 1-13). This is not the case for the Des-Gly 11 analog, whereby the single omission results in the repositioning of Lys 5 (denoted by a square) within the array of hydrophobic amino acids. Analogous to the effect of replacing Phe 5 of magainin 2 with a glutamate (see Figure 9A, lane 5) the endopeptidase exhibited decreased activity against this PGLa derivative. The Des-Lys 12 PGLa omission analog

Is a Deter-

(A) Cleavage reactions utilizing glutamate substitution analogs as substrates were separated by acid gel electrophoresis and visualized by Coomassie blue stain. The single letter amino acid code below each lane represents the residue replaced with a glutamate in that particular analog. Cleavage is detected by the appearance of the amino-terminal half-peptide as labeled on the right. (6) Space-filling axial and side representations of the first 12 residues of magainin summarizing the results depicted in the top panel. On the left, single residue glutamate substitutions that resulted in 0%-10% cleavage (relative to cleavage of native magainin peptide) are depicted in blue and are labeled Gly 1, Phe 5, Ala 9, Lys 11, Phe 12. Those substitutions that resulted in 25%-50% cleavage are depicted in green (lie 2, Gly 3, Lys 4, Ser 8) and substitutions resulting in 75%-106% cleavage relative to wild type are depicted in yellow (Leu 6, His 7, Gly 10). Percent cleavage of each substitution analog was determined visually by Coomassie blue-stained acid gels from several independent experiments, On the right are the native magainin (1-12) computer model representations as seen in Figure 7.

lacks the lysine on the carboxyl side of the scissile bond. Based on additional results reported below, we conclude that this analog is resistant to endoproteolysis because a basic, charged residue at this position is required for cleavage. Primary Sequence at the Cleavage Site Also Mediates Specificity The presence of a conserved lysine residue on the carboxy1 side of the cleaved bond in each natural peptide substrate suggests that primary sequence at the site of hydrolysis is a critical determinant of substrate specificity. To determine whether any basic residue can fulfill this criterion, the magainin 1 analog Kl 1 R, in which all lysines were replaced with arginines, was assayed (Figure 1F). The peptide effectively served as a substrate, demonstrating that any basic amino acid at the cleavage site is tolerated by the enzyme for hydrolysis. The possibility that the endopeptidase is even less specific and requires simply a charged residue, either basic or acidic, at this position was already addressed by the magainin Lys 11 glutamate

Endoproteolytic 549

Cleavage

of Amphipathic,

a-Helical

Peptides

-J Full length peptide 1

PGLS

Figure

10. Endopeptidase

2

3

4

5

Half peptides

6

(l-13)

Activity

against

PGLa

Omission

Analogs

Supports

the Deduced

Structural

Motif

(A) Acid gel electrophoresis of reaction products demonstrates cleavage of Des-Lys 5 and Des-Gly 11 PGLa analogs. Lanes run in the absence of enzyme and serve as control lanes of uncleaved, full-length peptides. Lanes 2,4, and 6 are reactions cleavage is identified by the generation of half-peptide. (B) Helical wheel projections of native PGLa (1-13) and the three single residue omission analogs assayed for cleavage in acids constituting the nonpolar face of each potential o helix are circled, the amino acids bracketing the scissile bond are the displaced lysine believed to affect cleavage of the Des-Gly 11 analog (Lys 5) is boxed.

1,3, and 5 are reactions including enzyme, and (A). Hydrophobic amino boxed and shaded, and

Cdl 550

substitution (see Figure 9A, lane 11). This derivative’s resistance to cleavage allows us to conclude that a basic amino acid is necessary for hydrolysis by the endopeptidase. Definitive proof that a lysine or arginine is required for endoproteolysis was established by assaying several peptide analogs lacking any charged residues at the site of hydrolysis. The PGLa omission analog Des-Lys 12, which results in Gly-lie at the scissile bond, was not susceptible to hydrolysis (see Figure lOA, lane 6) indicating the requirement of the lysine at this position. The magainin analog Kl 1 A, in which an alanine replaced the lysine at position 11, was assayed for cleavage and resisted attack, as did magainin analog Kll P, which contains a proline in place of Lys 11 (Figure 1F). Based on these data, we postulate that another determinant of substrate specificity is the presence of a basic residue on the carboxyl side of the scissile bond. Further characterization of the nature of the cleavage site entailed determining the limitations placed on the residue that comprises the amino side of the hydrolyzed bond. A survey of the residues present at this position in the substrates naturally synthesized by Xenopus laevis demonstrates the acceptance of lysine, glycine, and leucine. Cleavage of magainin 1 derivative GlOA revealed that an alanine at this site is also compatible with endoproteolysis (Figure 1 F), in accordance with the cleavage of PGLa analog Des-Gly 11 (Figure lOA, lane 4). Likewise, the magainin analog containing a substitution of glutamic acid for glycine (GlOE) at this position exhibited susceptibility to hydrolysis (Figure 9A, lane 10). The only tested amino acid found to be incompatible with cleavage by the endopeptidase is proline, which is consistent with our hypothesis that the endopeptidase recognizes a-helical secondary structure. The deleterious effect of introducing a proline into the substrate may be attributed to the ability of prolines to disrupt a helices. Inactive Substrates with Variations of the Structural Motif Still Demonstrate the Ability to Inhibit Endopeptidase Through the course of our extensive substrate specificity analyses, we came across several synthetic magainin analogs that did not serve as substrates for endoproteolysis. We decided to investigate whether these peptides resisted the action of theendopeptidaseat the level of initial binding or at the level of hydrolysis. One such example, the Desl-4 amino-terminal truncation analog of magainin 2 (Figure lB), was therefore utilized in inhibition assays designed to distinguish between these two possibilities. As illustrated in Figure 11, preincubation of the endopeptidase with the analog at a lo-fold molar excess effectively served to inhibit cleavage of magainin 2 (compare lanes 2 and 3). Furthermore, the Des-l-4 analog was found to inhibit endoproteolysis against PGLa, XPF, and CPF (data not shown), offering additional support for our claim that a single endopeptidase cleaves multiple peptide substrates. These data suggest that despite the loss of 4 residues from the amino terminus, rendering the peptide resistant to

cleavage by the endopeptidase, the structural parameters that direct the initial binding reaction are not compromised. Although unrelated to the magainins, melittin, an a-helical cytolytic peptide isolated from bee venom, exhibits ionophoric properties much like thexenopus antimicrobial peptides (Tosteson and Tosteson, 1981). These functional similarities most likely result from their shared structural features, and we therefore assessed the ability of the endopeptidase to treat melittin as a substrate (Figure 11, lane 4). Although this peptide was found to resist cleavage, examination of its primary sequence (Figure 1 G) may provide an explanation for this result. Melittin, despite its ability to adopt an amphipathic a-helical domain of at least 13 residues, does not have the requisite basic residue signaling cleavage at the proper location on the hydrophilic face of the helix. We also tested melittin as an inhibitor of enzymatic activity against the natural substrate magainin 2. Preincubation of the endopeptidase with a lo-fold molar excess of melittin (Figure 11, lane 6) served to inhibit cleavage, but only to a degree of approximately 25% relative to inhibition by magainin Des-l-4. We attribute this limited inhibitory capability to a lowered binding affinity (compared with magainin Des-l-4) for the endopeptidase, perhaps owing to the presence of a proline at residue 14. These results lead us to postulate that the maintenance of an uninterrupted a helix is necessary not only for cleavage by the endopeptidase but for efficient binding as well. Discussion We have purified to homogeneity a novel endopeptidase from the skin of Xenopus laevis that cleaves a family of antimicrobial peptides. This abundant 110 kd protein functions as a monomer and appears to be a metallopeptidase, based on its inhibition by metal chelating agents. Cleavage

Melittin

Mgn 2 [Des l-41 EnzymeI Inhibitor Substrate

Figure 11. Analysis

+ + -

+ +

+‘I+ + f

+ --

+ f

1

2

3

4

5

of Peptides

as Inhibitors

+ f f

’

6

of Endopeptidase

Activity

Acid gel of enzymatic assays designed to test the ability of magainin 2 Des-l -4 analog and melittin to inhibit endopeptidyl cleavage of native magainin P-amide. Control lanes 1 and 4 demonstrate that neither potential inhibitor serves as a substrate of the endopeptidase. Lanes 2 and 5 also serve as controls and show 100% cleavage of native magainin 2-amide in the absence of any inhibitor. Lanes 3 and 6 illustrate the results of incubating enzyme with a IO-fold molar excess of inhibitor prior to adding native substrate. Inhibition assays were conducted as described in Experimental Procedures.

Endoproteolytic 551

Cleavage

of Amphipathic.

a-Helical

Peptides

site analyses have confirmed that the endopeptidase is active against a single Xaa-Lys bond in each natural substrate examined, and the site of cleavage generated in vitro corresponds to those first reported in vivo (Giovannini et al., 1987). Through the utilization of numerous synthetic peptide analogs, the structural determinants of substrate specificity have been identified. Our results suggest that the endopeptidase recognizes an a-helical domain comprising at least 12 amino acids; a hydrophobic face; and a lysine residue on the hydrophilic face positioned precisely within the context of at least 4 nonpolar amino acids aligned along the hydrophobic face. The three-dimensional molecular models presented in Figure 8 demonstrate how sequences of negligible amino acid identity can all direct the adoption of this particular substrate conformation. Our data is consistent with a model in which the endopeptidase recognizes its peptide substrates as a-helical structures. Previous structural studies of the magainin peptides utilizing two-dimensional nuclear magnetic resonance, Raman, and circular dichroism spectroscopy have demonstrated their adoption of a helices in a phospholipid environment (Marion et al., 1988; Matsuzaki et al., 1989; Williams et al., 1990; Ducholier et al., 1989; Bechinger et al., 1991). Although these investigations revealed that magainins lack definable secondary structure in an aqueous solution and instead exist as random coils in the absence of protein and lipids, interaction with other proteins may serve to induce ordered structure. Analyses of this kind have been performed on peptides with high a-helical potential. Melittin, upon binding calmodulin as a 1:l complex, exhibits an increase in helical content from 5% to 70%, and similar conformational inductions have been reported for the interactions between calmodulin and numerous other amphipathic, a-helical peptides including 6-endorphin and mastoparan (Maulet and Cox, 1983; McDowell et al., 1985; O’Neil and DeGrado, 1990). The complex formation between p36 (annexin II) and its regulatory subunit pll was recently found to be mediated by the amino-terminal 12 residues of ~36. Structural studies of this peptide domain revealed its ability to adopt an amphipathic a helix upon binding pll and serve as further support for these interactions in nature (Johnsson et al., 1988; Becker et al., 1990). Synthetic peptide analogs designed to modify the secondary structural motif shared by the magainin peptides were tested against the endopeptidase, allowing us to identify particular determinants governing specificity. One such feature, revealed by the magainin glutamate substitution analogs, is substrate hydrophobicity. More specifically, one face of the amphipathic helix must be hydrophobic in order to be recognized as an optimal substrate. Although the requirement of a continuous registry of hydrophobic residues has never before been implicated in processing by endopeptidases, it has been observed for the binding of the amino-terminal portion of annexin II to pl 1, as well for the binding of basic, amphipathic peptides to calmodulin (Becker et al., 1990; O’Neil and DeGrado, 1990). We envision that a hydrophobic interaction be-

tween enzyme and substrate facilitates the initial binding reaction preceding hydrolysis. Inhibition of endoproteolysis by the magainin amino-terminal truncation analog Des-l-4, as well as an unrelated amphipathic, a-helical peptide, mastoparan (data not shown), provides direct evidence that binding of peptide to the enzyme is an individual component of the cleavage reaction. While these peptides do not manifest the complete structural motif necessary for hydrolysis and thus do not serve as substrates, their amphipathic, a-helical conformation is sufficient for binding the enzyme. In many proteolytic processing systems, substrate primary sequence at cleavage sites cannot entirely account for the selectivity of peptide bonds hydrolyzed by a given enzyme. For this reason, a large number of reports documenting endoprotease specificity implicate the role played by substrate secondary structure (Beinfeld et al., 1989; Gluschankof et al., 1988). Many investigators have recently begun to explore how changes in structure affect cleavage of substrates through the use of shortened synthetic analogs or precursor polypeptides modified by sitedirected mutagenesis of cDNAs (Brakch et al., 1989; Gomez et al., 1989; Docherty et al., 1989; Thorne and Thomas, 1990). Unfortunately, studies of this nature usually serve only to identify a specific residue at or nearby a cleavage site that influences substrate susceptibility, and the contributions made by the amino acid to the overall structure of the full-length substrate are not assessed. Our investigations focus on an endopeptidase active against physiological substrates that are small peptides and thus more amenable to manipulation. Synthesis of a highly informative variety of analogs allowed us to examine more rigorously the structural parameters governing endoproteolysis. Alternative strategies to probe the role of substrate structure in endoproteolysis have entailed examining the potential of putative recognition sites to adopt certain conformations. A statistical analysis revealing the high probability of 8 turns at cleavage sites characterized by paired basic residues served to direct attention to this particular structural motif (Rholam et al., 1986). This postulate has been directly challenged by experiments that introduced or deleted residues believed to influence the adoption of such a conformation within synthetic substrates (Brakch et al., 1989; Gomez et al., 1989). Most recently a computer algorithm designed to predict the occurrence of “omega loops” (long, unstructured loops) at known prohormone dibasic cleavage sites indicated that hydrolyzed bonds may indeed be associated with this conformational motif as well (Bek and Berry, 1990). Again however, the structural contributions made by the remaining regions of the polypeptide are often not addressed in predictive analyses of this sort and may remain untested owing to the difficulties encountered in manipulating large polypeptides. Precedence for an endoproteolytic enzyme that appears to recognize a structural motif rather than substrate primary sequence does exist. One exemplary model of sequence-independent processing is the action of peptidases involved in the cleavage of signal peptides of se-

C&Ii 552

creted proteins and the leader sequences that specify the targeting of mitochondrial proteins. Comprehensive studies by von Heijne and other investigators have established the role played by particular residues positioned near the

cleavage site in governing recognition by these endopeptidases (von Heijne, 1983; Duffaud and Inouye, 1988; Folz et al., 1988). However, amino acids far cleavage site both within the hydrophobic and the positively charged amino-terminal

upstream core region

of the domain charac-

teristic of signal peptides of secreted proteins have been found to profoundly influence processing as well. Amino acid substitutions proparathyroid

that distort the a-helical hormone signal peptide

potential or place

of prehydro-

philic residues into the conserved hydrophobic domain yielded poor substrates for the cleavage reaction catalyzed by the eukaryotic signal peptidase (Caulfield et al., 1989). Similarly, deletion of 4 residues from the amino terminus of yeast cytochrome oxidase subunit IV composing the mitochondrial targeting signal served to prevent cleavage at the wild-type site 25 amino acids downstream (Hurt et al., 1987). Several similarities between signal peptidase activity and the novel endopeptidase activity we describe in this article include the apparent sequenceindependent cleavage specificity and the requirement for an a-helical structural motif amino-terminal to the cleavage site. We hope to gain some insight into the mechanisms of recognition and hydrolysis utilized by our endopeptidase from the ongoing analyses conducted on signal peptidases and their cleavage specificity. The enzyme described in this report

is stored

in the

granular gland and secreted simultaneously with its substrates. Of interest will be the elucidation of how substrates and

enzyme

are compartmentalized

in the granular

gland

and the precise regulation of their interactions upon secretion. An understanding of the biological role of the endopeptidase

also

remains

to be determined.

Because

the

half-peptide products generated by endoproteolysis no longer retain antibiotic activity, the processing reaction may be regarded as an inactivating step. At the same time, however, several of the half-peptides have been shown to undergo subsequent carboxy-terminal amidation (Gibson et al., 1986), a modification characteristic of many biologically active peptides (Tatemoto and Mutt, 1978). Identification of modified half-peptides suggests that the endopeptidase may serve to liberate new hormones as well. It is our expectation that endopeptidases related to the Xenopus magaininase will be expressed in the mammalian nervous system, including both the central and peripheral divisions. A large body of work has shown that peptides found in frog hormone, tachykinin,

skin, such bombesin,

as thyrotropin-releasing and caerulein (cholecys-

tokinin), are present, as a rule, in the mammalian nervous system (Bevins and Zasloff, 1990). Since many neuropeptides exhibit a high propensity to adopt amphipathic a helices (Segrest et al., 1990; Kaiser and Kedzy, 1987), we speculate that relatives of this novel endopeptidase will play a role in neuropeptide processing in higher organisms. The availability of purified enzyme from Xenopus will facilitate these future studies.

Experimental

Procedures

Materials

Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, Wisconsin). Ammonium sulfate, glycerol, ammonium acetate, and phenylmethyisulfonyi fluoride were purchased from Bethesda Research Laboratories; “PAGE I” acryiamide, ieupeptin, and pepstatin were purchased from Boehringer Mannheim. Nonidet P-40, Triton X-100, phenanthroline, TPCK, E-64, phosphoramidon, and melittin were purchased from Sigma. Ampholytes were purchased from Bio-Rad. Mastoparan was purchased from Peninsula Laboratories, Inc. Peptide

Synthesis

Magainins 1 and 2, PGLa, XPF, CPF, and all derivatives tides were synthesized by the solid phase procedure described (Zasloff et al., 1966).

of such pepas previously

Purification

Approximately 50 g total of dorsal and ventral Xenopus skin was dissected from the anesthetized animals, weighed, and homogenized by a Polytron in 10 voi of 50 mM ammonium acetate, 15% (v/v) glycerol (pH 7.0) (buffer A). After centrifugation at 10,000 rpm for 40 min at 4%, the supernatant was collected and saturated with ammonium sulfate to 30%. Following precipitation and centrifugation, the supernatant was saturated to 60% with ammonium sulfate and again precipitated. The pellet was resuspended in 20 ml of buffer A and dialyzed extensively against the same buffer. The dialyzed sample was then subjected to preparative-scale, recycling free-flow isoelectric focusing on an RF-3 instrument (Protein Technologies, Inc., Tucson, Arizona). The cell was prefocused in 1% (v/v) pH 4-6 ampholytes, 15% (v/v) glycerol, and 0.1% (v/v) Triton X-100 for approximately 60 min at 1500 V. The sample was then focused for 120 min at a temperature of 7%-10%. Fractions (2.5 ml) were collected and assayed for enzymatic activity. All subsequent steps were carded out at 4%. Active fractions were pooled and concentrated by ultrafiltration using Centricon 30 (Amicon) tubes, then loaded onto a Sephacryl S-300 HR (Pharmacia) column (2.5 x 22 cm) equilibrated in 50 mM ammonium acetate, 0.1% (v/v) Nonidet P-40 (pH 7) (buffer 6). The sample was eluted in buffer B at a flow rate of 0.5 mllmin. Fractions (1.5 ml) were collected and monitored by UV absorbance at 260 nm. A subset of active fractions was again pooled after analysis of enzymatic activity and purity by denaturing gel electrophoresis. A Biogel HPT hydroxyapatite (Bio-Rad) column (1 x 5 cm) equilibrated in 50 mM sodium phosphate, 0.1% (v/v) NP-40 (pH 7) was next loaded with the pooled sample and washed with 10 column vol of the same buffer. Elution of the enzyme was achieved with a 25 ml linear gradient from 50 mM lo 500 mM sodium phosphate, 0.1% (v/v) NP-40 at a flow rate of 0.1 milmin. After analyzing the 0.5 ml fractions for enzyme activity, active fractions were pooled and concentrated by ultrafiltration. Glycerol gradients (12 ml) consisting of 15%-30% glycerol in 50 mM ammonium acetate were prepared in siliconized polyallomer (14 x 95 mm) tubes (Beckman). Pooled, concentrated enzyme sample from the hydroxyapatite column was applied to the top of the preformed gradient and centrifuged for 36 hr at 39,000 rpm in an SW40Ti rotor (Beckman). Fractions (0.25 ml) were collected from the bottom of the gradient tube and analyzed for enzymatic activity, as well as purity, by gel electrophoresis. Enzyme

Assays

Activity was monitored by incubating 0.1-10 ~1 of each chromatographic fraction with 50 wg of magainin P-amide substrate in a total volume of 100 ~1 for 1 hr at room temperature. Reaction buffer consisted of 20 mM sodium phosphate and 50 mM sodium chloride, final concentrations. After quenching the reaction with 0.5 voi of glacial acetic acid, a 15 PI aliquot of each reaction was analyzed by acid gel electrophoresis. One unit of enzyme activity is defined as the amount of enzyme required to convert 25 pg of magainin 2-amide substrate into half-peptide products under the above conditions. Substrate specificity analyses were carried out as described above, except that 50 pg of each substrate analog under investigation was substituted for magainin P-amide in the incubation.

Endoproteolytic 553

Cleavage

of Amphipathic,

a-Helical

Peptides

Inhibition assays were conducted by incubating the enzyme with each inhibitor at various concentrations for 1 hr at room temperature prior to addition of the magainin P-amide substrate. The reactions were allowed to proceed for an additional hour at room temperature before quenching as described above. Acid Polyacrylamide Gel Electrophoresis Acidgels(pH4)werepreparedandrunasoriginallydescribed(Gabriel, 1971) with several modifications. The gel solution consisted of a final concentration of 15% (v/v) acrylamide (acrylamide:bis-acrylamidel 37.5:1), 88 mM KOH, 3% (v/v) glacial acetic acid, 0.75% (v/v) Temed, 0.375% (v/v) ammonium persulfate. Before loading samples, 0.5 vol of glacial acetic acid was added (unless already added to quench enzyme assays as described), as well as 0.5 vol of 0.1% Pyronin Y loading dye. A Mini Protean gel apparatus (BieRad) was used, and gels were electrophoresed at 200 V in running buffer containing 0.035 M b-alanine and glacial acetic acid (2.45 ml/l) (pH 4.0). Gels were stained in 0.1% (w/v) Coomassie brilliant blue R-250 prepared in 50% methanol and destained in deionized water. SDS-PAGE SDS-PAGE was carried out following the method of Laemmli and gels were stained with silver nitrate. Protein molecular standards were purchased from Sio-Rad.

(1970), weight

HPLC and Amino Acid Analyses Enzyme reactions were prepared for HPLC analysis by termination with 1% trifluoroacetic acid at a final concentration of 0.1%. Reactions (100 ~1) were injected into a Beckman HPLC System Gold instrument and run on a Cl8 reverse-phase column (4.6 x 220 mm, Aquapore OD-300, Applied Biosystems, Foster City, California). A linear gradient of 30%-70%, buffer A: 0.1% trifluoroacetic acid in H20, buffer 8: 0.06% trifluoroacetic acid in acetonitrile, was established over a period of 45 min at a flow rate of 1 mllmin. Fractions containing peptide were lyophilized and resuspended in HPLC grade water. Portions of the resuspended sample were injected into an amino acid analyzer with automated hydrolysis (model 420/130, Applied Eiosystems), and numerical calculations for specific amino acids were determined using an Applied Biosystems 920A data analysis module. Protein Concentration Protein concentration was determined by either the Bio-Rad assay (adapted from the method of Bradford) or the Pierce BCA assay following the manufacturer’s instructions. Acknowledgments We wish to acknowledge David Christianson, William Lipscomb, Peng Loh, Ponzy Lu. and members of the Zasloff laboratory for helpful discussions. We especially thank Hernan Cuervo and Richard Houghten of the Torrey Pines Institute for their generous contribution of the magainin omission and glutamate substitution analogs, Jim Ostrem and Rodolfo Marquez of Protein Technologies, Inc. for assistance in applying preparative-scale isoelectric focusing to this investigation, and Howard Eck of the Nucleic Acid and Protein Core Facility at CHOP for amino acid analyses. This research was supported in part by grants from the Ben Franklin Partnership and the G. Harold and Leila Y. Mathers Charitable Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

April 4. 1991: revised

May 29, 1991

by solid-state

NMR spectroscopy.

J. Biomol.

NMR,

in press.

Becker, T., Weber, K., and Johnsson, N. (1990). Protein-protein recognition via short amphiphilic helices: a mutational analysisof the binding site of annexin II for ~11. EMBO J. 9, 4207-4213. Beinfeld, M. C.. Bourdais, J., Kuks, P., Morel, A., andcohen, P. (1989). Characterization of an endoprotease from rat small intestinal mucosal secretory granules which generates somatostatin-26 from prosomatostatin by cleavage after a single arginine residue. J. Biol. Chem. 264, 4460-4465. Bek, E., and Berry, R. (1990). Prohormonal cleavage ated with omega loops. Biochemistry 29, 178-183. Bevins, C. L., and Zasloff, M. (1990). Rev. Biochem. 59, 395-414. Boman, H. G. (1991). Antibacterial in immunity. Cell 65, 205-207.

Peptides

peptides:

sites are associ-

from frog skin. Annu.

key components

needed

Boman, H. G., Boman, I. A., Andreu. D., Li, Z., Merrifield, R. B., Schlenstedt. G., and Zimmermann, R. (1989). Chemical synthesis and enzymic processing of precursor forms of cecropins A and B. J. Biol. Chem. 264, 5652-5860. Brakch, N., Boussetta, H., Rholam, M., and Cohen, P. (1989). Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones. J. Biol. Chem. 264, 15912-15916. Caulfield, M. P., Duong, L. T., Baker, R. K., Rosenblatt, M., and Lively, M. 0. (1989). Synthetic substrate for eucaryotic signal peptidase. J. Biol. Chem. 264, 15613-15817. Darby, N. J., and Smyth, mone processing. Biosci.

D. G. (1990). Endopeptidases Rep. 70, I-13.

and prohor-

Darby, N. J., Lackey, D. B., and Smyth, D. G. (1991). Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis. Eur. J. Biochem. 195, 65-70. Docherty, K., Rhodes, C. J., Taylor, N. A., Shennan, K. I. J., and Hutton, J. C. (1989). Proinsulin endopeptidase substrate specificities defined by site-directed mutagenesisof proinsulin. J. Biol. Chem. 264, 18335-l 6339. Dockray, G. J.. and Hopkins, C. R. (1975). Caerulein secretions dermal glands in Xenopus laevis. J. Cell Bioi. 64, 724-733.

by

Duclohier, H., Molle, G., and Spach, G. (1989). Antimicrobial peptide magainin 1 from Xenopus skin forms anion-permeable channels in planar lipid bilayers. Biophys. J. 56, 1017-1021. Duffaud, G., and Inouye, M. (1988). Signal peptidases recognize a structural feature at the cleavage site of secretory proteins. J. Biol. Chem. 263, 10224-10228. Folz, R. J., Nothwehr, S. F., and Gordon, J. I. (1988). SubstratespecificIty of eucaryotic signal peptidase. J. Biol. Chem. 263, 2070-2078. Gabriel, 0. (1971). Analytical 22, 565-576.

disc gel electrophoresis.

Meth. Enzymol.

Gibson, B. W., Poulter, L., Williams, D. H., and Maggio, J. E. (1986). Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis. J. Biol. Chem. 261, 53415349. Giovannini, M. G.. Poulter, L.. Gibson, B. W., and Williams, D. H. (1987). Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones. Biochem. J. 243, 113-120. Gluschankof, P., and Cohen, P. (1987). Proteolytic enzymes in the post-translational processing of polypeptide hormone precursors. Neurochem. Res. 72, 951-958. Gluschankof, P., Gomez, S., Lepage, A., Creminon, C., Nyberg, F., Terenius, L., and Cohen, P. (1988). Role of peptide substrate structure in the selective processing of peptide prohormones at basic amino acid pairs by endoproteases. FEBS Lett. 234, 149-152.

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Bechmger, B., Kim, Y., Chirlian, L. E., Gesell, J., Neumann, J.-M., Montal, M., Tomich, J., Zasloff, M., and Opella, S. J. (1991). Orientationsofamphipathic helical peptides in membranebilayersdetermined

Hurt, E. C., Allison, D. S., Muller, U., and Schatz, G. (1987). Aminoterminal deletions in the presequence of an imported mitochondrial protein block the targeting function and proteolytic cleavage of the

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E. T., and Kezdy, F. J. (1967). Peptides with affinity Annu. Rev. Biophys. Chem. 76. 562-561.

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