Comp. Bioehem. Physiol., 1972, VoL 43B, pp. 419 to 424. Pergamon Press. Printed in Great Britain

THE EVOLUTION OF ENDOPEPTIDASES--XIV. NON-TRYPTIC CLEAVAGE SPECIFICITY OF A

BAEE-HYDROLYZING ENZYME (fl-PROTEASE) FROM TENEBRIO MOLITOR

ROBERT Z W I L L I N G * , IVAN M E D U G O R A C and K L A U S M E L L A

Department of Chemistry, Ruhr-University Boehum, 463 Bochum, Germany

(Received 9 February 1972)

Abstract--1. fl-Protease, an endopeptidase of molecular weight 25,000 from the digestive tract of adult Tenebrio molitor beetles, has been purified to an electrophoretic pure state. The preparation was free of exopeptidase as well as of endopeptidase activities of different cleavage specificity.

2. fl-Protease is inhibited by all natural trypsin inhibitors and splits the trypsin substrates N~-benzoyl-arginine ethyl ester and poly-lysine. It therefore had to be decided by the use of more complex peptide substrates whether the cleavage specificity is really tryptic.

3. When tried with chain B of oxidized insulin and a dodeca-peptide, the cleavage specificity of fl-protease is shown to be clearly different from that of mammalian trypsin.

4. The possible relationships of fl-protease to the group of trypsin-like enzymes are briefly discussed.

INTRODUCTION

DURING our investigations of the molecular evolution of endopeptidases from invertebrate animals special interest was given to possible precursors of mammalian trypsin. In several species of decapod crayfish we demonstrated the occurrence of "crayfish trypsin", which shares all decisive properties with vertebrate trypsin, including the most restricted cleavage specificity, the inhibition specificity against all natural and synthetic trypsin inhibitors tried and the characteristic -asp-ser -g ly- sequence of pancreatic serine-proteases (Pfleiderer et al., 1967; Zwilling et al., 1969; Tom~i~ek et al., 1970). In insects (honey bee) we encountered a trypsin-like enzyme, which displayed exactly the same cleavage specificity as bovine trypsin when assayed on complex polypeptide substrates (Giebel et al., 1971).

On the other hand, in several other invertebrate species (for example, Sepia ofl~cinalis, Limulus polyphemus), we failed to detect enzymes with tryptic cleavage specificity.

Earlier we also discussed to what degree cleavage of synthetic trypsin substrates might constitute a proof for "tryptic cleavage specificity" (Zwilling et al., 1969).

* Present address: Wiss. Rat Dr. R. Zwilling, Zoologisches Institut der Universit~it Heidelberg, 69 Heidelberg, Berliner Str. 15, Germany.

419

420 ROBERT ZWILLING, IVAN MEDUGORAC AND KLAUS MELLA

One has to consider that the substrate N~-benzoyl-arginine ethyl ester (BAEE), widely used to demonstrate "trypsin activity", is split as well by such plant SH- proteases as papain and bromelain, which are not related to the trypsin family.

All this had to bc taken into account when we isolated from Tenebrio molitor an enzyme fraction capable of hydrolyzing BAEE and being inhibited by all natural trypsin inhibitors tested (~-protease (Zwilling, 1968).

Furthermore, Applebaum et al. (1964) in a previous study have demonstrated an activity in T. molitor splitting poly-lysine and carbobenzoxy-tyrosine nitro- phenyl ester and being inhibited by crystalline soybean trypsin inhibitor, which they termed "Tenebrio trypsin".

We would now like to point out that this endopepfidase from Tenebrio-- though splitting synthetic trypsin substrates and polylysine--does not possess a real tryptic cleavage specificity when tried on complex po]ypcptide substrates such as the B chain of oxidized insulin. Therefore, the designation "trypsin" should not be applied to it.

MATERIAI,S AND METt tOI)S Materials and methods were those given in detail in our previous communications

(see Zwilling et al., 1969; Zwilling, 1968). The digestive juice of Tenebrio midgut was obtained from about 6000 adult beetles

(Pfleiderer & Zwilling, 1966) and purified by gelfiltration, anion exchange chromatography and repetition of the gelfiltration step. As fl-protease in its natural condition forms aggre- gates of a molecular weight of about 60,000 (Zwilling, 1968), by the first gelfiltration step it could easily be separated from all other accompanying endopeptidases (tool. wt. about 25,000) which do not show this behaviour. The fl-protease aggregates are suspended by the anion exchange step.

The whole purification procedure was based on the BAEE-hydrolyzing activity of fl-protease.

A qualitative amino acid analysis from the hydrolysis products which we received from the substrates chain B of insulin and a dodeca-peptide was performed in a Tecbnicon Auto- Analyzer in order to establish the exact cleavage specificity of ~-protease.

RESULTS Purity of fl-protease

T o assure the high degree of puri ty indispensable for the s tudy of proteolytic cleavage specificity, /3-protease was finally purified by an electrophoretic pro- cedure.

As shown in Fig. 1, the purified fraction was submit ted to cellulose acetate foil electrophoresis. T h e foils were cut longitudinally and while one- third was stained for proteins, the remaining two-thirds were retained for elution. T h e unstained port ion was subdivided into fractions as shown and BAEE-hydrolyzing as well as caseinolytic activities were measured for each fraction. As can be seen f rom Fig. 1, 3-protease activity, distinguishable by its BAEE-spl i t t ing activity, was concentrated under these conditions in fraction 9.

T H E E V O L U T I O N O F E N D O P E P T I D A S E S - - X I V 421

At the same time the small caseinolytic activity, appearing in fraction 4 and having escaped the previous purification steps, could be eliminated completely.

With the exception of the intense spot in fraction 9 (fl-protease) no other protein bands were detectable by amino black staining. We also chose the above procedure to prove that it was obviously the BAEE-hydrolyzing activity from which we obtained an unexpected cleavage pattern when it was tried on polypeptide substrates. During the following investigations fl-protease exclusively derived from fraction 9 was used.

(+) Fract. No

,%

,8 - proteose- ( '

,~,~ ,) ~...~_~ ~,,"~_o-°'~ , ~.x,h:,

l-llJ-m-m-J-Ii-I Sta rt

-o4oo5 ,2oo .~ _> =

-- 0300 --u D'800 o

D.20~ "~ i i

o ~.100 ~

I.d h i

Cl2

Eluted -I

Stained {_)

FIO. 1. Electrophoresis of purified fl-protease f rom T. molitor on cellulose acetate foils (for details see text). Buffer: barbital 0"024 M, p H 8"6; 1 h r at 150 V and

2 mA/foil.

Cleavage specificity Of the synthetic substrates tried, N-acetyl-L-tyrosine ethyl ester (ATEE),

N-acetyl-L-phenylalanine-naphthylester (APNE), N~-benzoyl-L-arginine naphthyl- amide (BANA) and glutaryl-L-phenylalanine-2-naphthylamid (Glupna) were not hydrolyzed by/3-protease.

Purified/3-protease also did not split hippuryl-L-lysine, which proves that no traces of an originally present carboxypeptidase activity were left in the preparation.

On the other hand, N~-benzoyl-L-arginine ethyl ester (BAEE), a substrate considered to indicate trypsin activity, is readily attacked. We can also confirm a similar observation of Applebaum et al. (1964) that poly-lysine is a good substrate for/3-protease. But the fact that BANA, another good trypsin substrate, is not hydrolyzed by/3-protease as we have already mentioned above, was a hint that there might be no strictly tryptic cleavage specificity.

422 ROBERT ZWILLING, IVAN MEDUGORAC AND KLAUS MELLA

When chain B of the oxidized bovine insulin was incubated with pure /3- protease, the electrophoretic separation of the hydrolysis products resulted in four bands, none of which were identical to the three bands produced by the action of bovine trypsin (Fig. 2). After ehtion of each peptide band produced by ~-protease, the amino acid composition of the separated peptides was established.* Thus, from the known covalent structure of chain B the composition of the single pep- tides A-D as well as the points of cleavage were derived (Fig. 2).

(-)

Peptide C: His s-Tyr t6 C - - ]

Peptide B: Phet-Glu 4 [ ]

Peptide A: Leul~'-Phe 5 [ )

~ _ ~ Gly :~-Lys' '

Phe i_ Arq ::

r TT Start J t i ___J (+) ,~-protease- trypsin -

catalyzed

FIC. 2. Hydrolysis products of incubation of chain i} of oxidized insulin with 8-protease (I) and trypsin (lI), separated by paper electrophoresis. Two mg chain B + 0.5 ml buffered enzyme solution, incubated for 1 hr at 35°C and pH 8"0.

The cleavage pattern of fl-protease is completely different from that of mam- malian trypsin (Fig. 3). The bonds arg~2/gly 2~ and lys~9/ala ~°, characteristically hydrolyzed by the trypsins, are not attacked at all by ~-protease. The incubation of chain B with/3-protease also does not yield any free alanin, which is liberated by the action of trypsin.

To assure these findings, we also tried the dodeca-peptide derived from the active centre of pig muscle lactate dehydrogenase as a substrate. In this sequence,

* Additionally each N-terminal residue of the eluted peptides was confirmed by Edman degradation.

THE EVOLUTION OF ENDOPEPTIDASES--XIV 423

which is completely resistant to trypsin, a leu/asp-bond was readily split by /~- protease (Fig. 3).

I . ChainB of oxidized insulin

H.Phel -Val2 -As3p :-Glu4 -His5 -Leu6 --C~'s-GlY8 -Ser9 --HiSlo -LeUll -Vail2 -Glul3 -Alal4 -LeUl5 -

I I l Peptide B Peptide C

,/~- profease Cfh Chl Tr~ ch~ C¢ Tr~ 'Tyr LLeu --VoI --Cys -GIy -61u -Arg LGly -Phe - Phe-LTyr .-Thr -Pro -Lys : -A la -OH

t6 17 t8 19 20 21 22 23 24 25 26 27 28 29 30

Peptide A Peptide D ]~- protease p - protease

Z.Dodeca-peptide ( from porc muscle lactate dehydrogenase , active center)

Vol - Ile -Gly -Ser -Gly --Cys --Asn --Leu -Asp - Ser -- AIo--Arg

/£- protease

FIG. 3. Points of cleavage of fl-protease on chain B of oxidized insulin (D and on a dodeca-peptide (II). For comparison the points of cleavage of trypsin and of

chymotrypsin are also indicated.

DISCUSSION As shown during the present investigation in adult 2". molitor beetles, no

proteolytic activity could be detected which possesses a tryptic cleavage specificity when tried on peptide substrates.

The fact that/~-protease splits the synthetic trypsin-substrate BAEE as well as poly-lysine and is inhibited by all naturally occurring trypsin inhibitors only demonstrates once more that these are not definite proofs for trypsin specificity.

Though Applebaum et al. (1964) prepared their Tenebrio trypsin from Tenebrio larvae it is probable that this protease also, possessing all characteristics of/~- protease from adult beetles, does not exhibit a cleavage specificity confined strictly to basic amino acid residues when tried on peptide substrates. Whether the two forms of/~-protease from the adult and larvae stages are identical or not is at present being studied by us.

Recently, by comparison of primary structures, it was shown that proteases of the trypsin family can be traced back to the organizational level of bacteria (Olson et al., 1970). Also, in several invertebrate species proteases with a cleavage speci- ficity identical to that of vertebrate trypsin could be demonstrated (ZwiUing et al., 1969). It remains an open question why in other species like Tenebrio no such cleavage specificity can be detected. It therefore could turn out to be that proteases of the type of fl-protease form Tenebrio with certain similarities to pancreatic

424 ROBERT ZWILLING, IVAN MEDUGORAC AND KLAUS MELLA

serine proteases, const i tute this "miss ing l ink", having changed by muta t ion certain propert ies of their catalytic site bu t having conserved in their p r imary s t ruc ture features homologue to the t ryps in / ehymot ryps in g roup of proteases.

Acknowledgements--The present studies were supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg.

REFERENCES

APPLEBAUM S. W., BInn Y., ttAI~eARZ I. & BONDI A. (1964) Comparative studies on proteo- lytic enzymes of Tenebrio molitor L. Comp. Bioehem. Physiol. l l , 85-103.

(;IEBEI. W., ZWILLING R. & PFLEIDERER G. (1971) The evolution of endopeptidases--XlI. The proteolytic enzymes of the honey bee (Apis mellifiea L.). Comp. Bioehem. Physiol. 38B, 197-210.

()I.SON hl. O. J., NAGABIIUSttAN N., DZWINIEL NI., SMILLIE I,. B. & WIIITAKER D. R. (1970) Primary structure of ~-lytic proteasc: a bacterial homologue of the pancreatic serine proteases. Nature, Lond. 228, 438-442.

PI~I.EIImRER G. & ZWlLLIN{; R. (1966) l~ber eine im physiologischen Bereich native Proteine hydrolysierende Endopeptidase aus dem Darm von Tenebrio molitor. Reinigung und Charakterisierung des Enzyms. Bioehem. Z. 344, 127-140.

I)I"I.EII)EIlFJI G., Z'WIIAANG R. ~'¢ S()NNEBORN }I.-1{. (1967) Zur Evolution der Endopepti- dasen 1II. Eine Protease vom Molekulargewicht 11000 und eine trypsiniihnliche Fraktion aus Astacusfluviatilis Fabr. Z. Physiol. Chem. 348, 519-524.

'I'oMASEK V., Sol~M F., ZWILLINC R. & Pvlam)F.Itgl~ G. (1970) Serine-containing active center of the trypsin-like protcase of crayfish ANtaeus leptodactylus. FEBS Lett. 6, 229-231.

ZWILLING R. (1968) Zur Evolution der Endopeptidasen--IV. a- und fl-Protease aus Tenebrio molitor. Z. Physiol. Chem. 349, 326-332.

ZWILLING R., PFLEIDERER G., SONNEBORN H.-H., KRAFT V. & STUCKY I. (1969) The evolution of endopeptidases--V. Common and different traits of bovine and crayfish trypsin. Comp. Biochem. Physiol. 28, 1275-1287.

Key Word Index--Endopeptidase ; ]~-protease ; BAEE ; Tenebrio molitor ; trypsin.