Proteolytic enzymes from the mouse submaxillary gland: A partial sequence and demonstration of spontaneous cleavages

ARCHIVES Vol.

OF BIOCHEMISTRY 209,

No.

1, June,

AND

BIOPHYSICS

pp. 57-62, 1981

Proteolytic Enzymes from the Mouse Submaxillary Sequence and Demonstration of Spontaneous ISAAC

Gland: A Partial Cleavages1y2

SCHENKEIN, EDWARD C. FRANKLIN,* AND BLAS FRANGIONEt

New York University Medica.l Center, Irvin&m House Institute, Departments of Microbiology, *Medicine, and +Pathologg, 550First Avenue, New York, New York 10016 Received October 21, 1980 The mouse submaxillary proteases (A and D), whose isolation and properties were previously described by us, hydrolyze only arginyl bonds in proteins. The sequence of 40 residues from the amino terminus has been determined, Its structure shows a striking (> 50%) homology with other enzymes of the serine group (e.g., trypsin, prothrombin, plasminogen, hall&rein). A single peptide labeled with dilsopropylfluoroE3*P]phosphate had a composition virtually identical to that found in the above proteases, and one active site per molecule was confirmed. The enzymes are very susceptible to spontaneous fragmentation which leads to two cleavages. The first converts enzyme A to D with loss of a small peptide. The second can only be demonstrated after reduction since the fragments appear to be joined by a disulflde bridge. The two resulting fragments with molecular weights of 14,000 and 11,000, respectively, are inactive. minants but show a marked difference in kinetic behavior toward substrates like TAME or BAME. A had normal kinetics; D showed sigmoidal kinetics and had an unusual sensitivity toward ions like Fez+ or Fe3+. The sigmoidal kinetics could be abolished by the addition of certain chelators of metal ions, such as glycine and &OH-quinoline and other a-amino acids. For the latter, no stereospeciflcity was observed (3). This report deals with the observation that enzyme A becomes enzyme D as a result of a spontaneous cleavage and that hydrolysis of an additional peptide bond in the enzyme molecule leads to fragmentation upon reduction. The latter point of cleavage must therefore be flanked by a disulfide bridge. The fragments are devoid of enzymatic activity. We also present the sequence of the first 40 residues from the amino terminus and the amino acid composition of a tryptic peptide isolated after reaction of the enzyme with [32P]DFP. There is a striking homology of its structure with those of several other proteolytic enzymes of the serine group, a finding consistent with the inhibition of both enzymes A and D by DFP which was shown earlier (3, 4).

We have reported previously on the physical and kinetic characteristics, as well as the singular substrate specificity for arginine, of two proteolytic enzymes from the male mouse submandibular gland (MSG)3 (l-4), one of which (enzyme D) is identical to the EGF carrier protein described by Taylor et al. in 1974 (5). The salient data can be summarized as follows: With proteins or homopolymers of amino acids, e.g., polyarginine or polylysine, the only cleavage occurs at carboxy-terminal bonds of arginine, but not of lysine. Both TAME and BAME are substrates; with TAME the K,‘s were 8 x 10m4M for enzyme A and 3 x 10m4 for enzyme D at 25°C. Maximum specific activity at pH 8.0 with TAME was 2500-3000 PM mine1 mg-’ with A, and 400-600 pM min-’ mg-’ with D. The enzymes referred to as A and D are closely related in terms of molecular weight, amino acid composition, and immunological deter’ Supported by USPHS Research Grant AM 01431. * This paper is Part III of a series. 3 Abbreviations used: MSG, mouse submandibular gland; TAME, tosyl-arginine-methyl-ester; BAME, benzoyl-arginine-methyl ester; DFP, Diisopropylfluorophosphate; NGF, nerve growth factor; EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; DTT, dithiothreitol. 57

ooO3-9861/81/070067-~02.00/0

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

58

SCHENKEIN,

FRANKLIN,

AND FRANGIONE

matography on polyamide plates (7), identification of the free amino acid residue after “back hydrolysis” in 0.25 ml of 6 N HCl + 5-10~1 of 5% SnCl, in w-0 (8) and high-pressure liquid chromatography using a Waters high-pressure liquid chromatograph Model ALCIGPC-204 with a 3.9 mm x 30 cm PBondapak Cl8 column (Waters Associates, Milford, Mass.) (9). Formation of enzywm D from A. This is accomplished by allowing enzyme A to stand at room temperature for 24-48 h at a concentration of 5-10 mg/ml in 0.01 M Tris buffer at pH 8.0. The mixture of enzymes A and D present at the end of the period of standing is then separated by gel filtration on Sephadex G-50. RESULTS FIG. 1. Composite of SDS-polyacrylamide gel electrophoresis runs. Lane A: Aliquot of mixture of A and D. Lane B: Aliquot of enzyme D isolated from mixture of A and D. Lane C: Aliquot of enzyme D under reducing conditions. Bands of M, 25,000, 14,000, and 11,000 are seen. Molecular weight markers (not shown) used were chymotrypsin, 26,ooO; lysozyme, 15,ooO, and RNase, 13,400. Mobility of D is less due to reduction. Lane D: Aliquot of the M, 14,000 fragment obtained by reduction of enzyme. D. Lane E: Aliquot of the M, 11,000 fragment obtained by reduction of enzyme D. MATERIALS

AND METHODS

Zeolation of pure enzyme A. In an earlier publication we described the isolation of pure A either by preparative acrylamide gel electrophoresis or by allowing the supernatant of the 62-70% ethanol precipitation step (see Refs. (2, 3) to stand for 72 h at -20°C and centrifuging the precipitate which formed. In this way some 200-300 mg of pure A can be obtained from a batch of 1000 submaxillary glands of adult male mice (purchased from Pell Freeze, Rogers, Ark.). Spontaneous formation of D from A was avoided by solvating pure A in acidic solutions, e.g., 0.1 M acetate buffer, pH 4.25 (see below). At this pH, the enzyme is inactive, even with synthetic substrates like TAME or BAME (3). Analytical procedures. Assay of enzymatic activity, high-voltage electrophoresis, paper chromatography, elution of peptides from paper, reduction and alkylation, SDS-polyacrylamide gel electrophoresis, and quantitative amino acid analysis and protein determinations have all been described elsewhere (3, 4). Amino-terminal analysis was performed by the manual Edman degradation (6). Amino acid analysis was used to identify the cleaved derivatives. Automated amino acid sequence analysis was performed with a Beckman 890C sequencer, using 0.1 M quadrol buffer. Identification of the phenylthiohydantoin derivatives was done by at least two and usually three methods. They are: two-dimensional thin-layer chro-

(1) Conversion

of Enzyme A to D

Fifty milligrams of A was allowed to stand overnight in 0.01 M Tris-HCl (pH 8.0), 1 x lo+ M sodium aside at room temperature. The protein gave a single band in SDS-gel electrophoresis, showed simple kinetics with TAME as substrate (see introduction), and had the appropriate immunoelectrophoretic mobility (l-3). After 16 h, the solution was acidified in the pHstat to pH 4.0 with 0.1 N HCl and suitable aliquots were subjected to preparative and analytical gel electrophoresis, immunoelectrophoresis, and a study of the rate of hydrolysis of TAME. The results were in accord with the concept that a fragment of M, 3000-5000 had been cleaved from A, forming a mixture of A and D (Fig. 1, lane A). The mixture was then subjected to molecular sieving on a column of Sephadex G-50 superfine (Fig. 2). Materials from peak 1 (tubes 210-225) and peak 2 (tubes 235250) were rerun under the same conditions and in this manner highly purified A and D were obtained. The fragment of M, 30005000 eluted near the retention volume of the first column (tubes 300-320) and has not been studied in any detail. SDS-gel electrophoresis of enzyme D, followed by staining with Coomassie blue, revealed less than 5% cross-contamination (Fig. 1, lane B). (.) Evidence for Additional

Digestion

Examination of the Coomassie blue staining pattern obtained upon running en-

59

MOUSESUBMAXILLARYPROTEASES

63 0502 025-

200 220 240 Fraction

number

FIG. 2. Molecular sieving on Sephadex G-50 superfine (column dimensions 2.5 x 250 cm) of 50 mg of enzyme A that had been converted to mixture of enzymes A and D (see Results). Fractions were 15 ml.‘Flow rate was 10 ml/h.

zyme D in SDS-gel electrophoresis under reducing conditions (see Materials and Methods) revealed two bands of iV, 14,000 and 11,000, respectively, in addition to the one with the molecular weight of enzyme D (i.e., 25,000) (Fig. 1, lane C). Only a single band was present when the material was run in SDS-gel electrophoresis under nonreducing conditions. These three components were separated by filtration of enzyme D after reduction in DTT in the presence of 8 M urea followed by alkylation with [14C]iodoacetic acid through a column of Sephadex G-50 in 10% formic acid (Fig. 3). All peaks had radioactivity and none had enzymatic activity with TAME as test substrate. Analytical SDS-gel electrophoresis indicated that peak 1 corresponded to enzyme D and peaks 3 and 4 to the M, 14,000 and 11,000 fragments, respectively. Peak 2 was not studied (Fig. 1, lanes D and E). A reasonable interpretation of the data in terms of structure is given in the discussion.

(4) Reaction

of Enzyme

A with r2PjLlFP

As indicated under Materials and Methods, 50 mg of enzyme A was reacted with r2P]DFP. Suitable aliquots were assayed for the hydrolysis of TAME as a function of time until total inhibition was observed; the calculated stoichiometry confirmed earlier published data (3). The data from pa-stat titration indicated that the reaction followed a first-order equation. The bimolecular reaction constants K (18.7 and 20.1 M-’ min-’ at pH 7 and 25°C) for two experiments are in agreement with

(3) Sequence Studies The sequence of the first 40 residues obtained with intact enzyme A and the fragment of M, 14,000 are given in Fig. 4. Since the fragment of IV, 14,000 was shown to have the same sequence as the enzyme, its position in the intact molecule is established. Comparison of the sequences at the ammo terminus of several other serine proteases with that of enzyme A reveals a significant homology (Fig. 4).

Fraction

rumber

FIG. 3. Separation on Sephadex G-50 fine of fragments obtained from enzyme D after reduction with D’IT in 8 M urea followed by alkylation with [W]iodoacetic acid in 10% formic acid. Flow rate and fraction size as in Fig. 1. Vertical bars indicate radioactivity in a l-ml aliquot of the pooled fraction. (1) Nonfragmented enzyme D, 18,000 cpm; (2) not studied, 2000 cpm; (3) fragment of M, 14,000,8000 cpm; (4) fragment of kf, 11,000, 15,000 epm.

(cow)

FIG. 4. Comparison of the amino-terminal sequences of enzyme A and the M, 14,000 (14K) fraction with those of other selected serine proteases taken from Dayhoff (10). Gaps - are introduced to maximize homology. Residues which are identical to enzyme A in at least four of the five enzymes are enclosed in boxes. Some heterogeneity is seen in enzyme A and in the 14K fragment. ( ) Unidentified residue.

Trypsin

14K Fragment

MOUSE SUBMAXILLARY TABLE AMINO

ACID

and thrombin (10) is shown in Table IB. There is a very close correspondence except for a lower than expected yield of cysteine which can be accounted by its low recovery in the unoxidized state. Since the release of valine is slow, the peptide probably contains two residues of valine.

IA OF A [S*P]DFP LABELED PEPTIDE

COMPOSITION TRYPTIC

Residue

Number of residues

ASP CYS Glu GUY Ser

2.3 0.9 1.3 4.5 0.9 0.9 1.3 1.1

PI-0

Val LYS

DISCUSSION

The structural data presented in this paper permit the placing of enzyme A into the group of serine proteases and point to spontaneous cleavage as the cause for the observed fragment formation. The structural relationships of A and D and some of the larger spontaneous proteolytic fragments indicate a marked propensity for further digestion which can be signiflcantly reduced at pH 4, thus facilitating isolation of the intact enzyme. Figure 5 and its legend suggest one of the possible routes of fragmentation. Based on the identity of the amino acid sequence of the l& 14,000 fragment and the intact enzyme, it is possible to position that fragment at the amino-terminal end of the molecule. The fragment of J4, 11,000 is probably adjacent to it, but since its sequence and that of the fragment of M, 3000-5000 are not yet known, the possibility that the M, 30005000 fragment is located between the two large fragments cannot be excluded. However, it appears less likely since its formation in this fashion would require still another endoproteolytic cleavage. Enzymes of the serine class show multiple intrachain disulflde bonds and the linkage proposed in the sequence is a common feature (10). The possible physiological role of both of the enzymes and the original small frag-

the data published in Ref. (3). After exhaustive dialysis and two separations on a Sephadex G-25 column, the material eluting in the void volume was digested with N--p-tosyl-L-lysin chloromethyl ketone HCl-trypsin (2 x 0.5 mg) at pH 8.0 in a Radiometer Copenhagen pH-stat (2). The digest was lyophilized and a small aliquot subjected to chromatography and high-voltage electrophoresis on Whatman 3MM paper as indicated under Materials and Methods. One major and several minor peptides stained with ninhydrin were shown to be radioactive by autoradiography. The peptide with the bulk of the radioactivity was eluted from an unstained paper with formic acid and subjected to acid hydrolysis and amino acid analysis. The composition is given in Table IA. It contained one mole of serine and the remaining residues gave a near-perfect fit with those of related proteins with activesite serine DFP adducts. The probable amino acid sequence based on homology to the region flanking serine 195 in trypsin TABLE PROPOSED

ALIGNMENT

BASED

61

PROTEASES

ON HOMOLCGY 1% IN THROMBIN

IB

TO THE RESIDUES FLANKING AND TRWSIN (lo)

THE ACTIVE

SITE

Residue 189 190 191 192 193 194 Thrombin Trypsin (bovine) Enzyme A

195 196 197 198 199 200

201

202

Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-ValMet -Lys Asp-Ser-Cys-Gln-Gly-Asp-Ser-Gly-Gly-Pro-Val-ValCys -Ser Asp, Gly, Cys, GIu, Gly, Asp, Ser, Gly, Gly, Pro, Val, Val, (Cys), Lys

62

SCHENKEIN, A -----s first &wage

-

FRANKLIN,

A*D p----* F-----F

_ %PlM

AND FRANGIONE

likrein system or the complement cascade) play a physiological role. It is of interest to note that similar processing is involved in the conversion of prothrombin to thrombin, plasminogen to plasmin, and prekallikrein to kallikrein. REFERENCES

5. One possible route of fragmentation. Purified enzyme A is cleaved with the loss of a smah fragment to give a mixture of A and D (M, 28,999 and 25,999, respectively). Enzyme D can undergo a second cleavage within one of the disuI6de loops resulting in the formation of two fragments of M, 11,999 and 14,999 after reduction. FIG.

ment, all of which must exist in viva since both A and D appear to be present intracellularly, is unknown. In elegant experiments, Von Deimling and his group (11, 12) and Barka and co-workers (13, 14) have demonstrated the intracellular localization of the granules that contain the enzymes, using novel techniques for detection of protease activity in SDS-polyacrylamide gels as well as fluorescent antisera. Support for their suggestion of the existence of genetic polymorphism is provided by some of the sequence heterogeneity shown in Fig. 4. The enormous quantity of enzymes A and D in adult male mice (500-600 mg per 1006 glands, i.e., 506 animals) and the fact that either biosynthesis or storage is under androgen control cannot easily be explained. The same appears to hold true for NGF and EGF which have been studied for several decades. The possibility arises that complex cascade phenomena occur in the submandibular gland, and that the smaller fragments produced (not unlike the kal-

1. SCHENKEIN, I., BOSEMAN, M., TOKARSKY, E., FISHMAN, L., AND LEVY, M. (1969) Biochem. Biophys. Res. Commun. 36, 156. 2. BOESMAN, M., LEVY, M., AND SCHENKEIN, I. (1976) Arch. Biochem. Biophys. 175, 463. 3. SCHENKEIN, I., LEW, M., FRANKLIN, E. C., AND FRANGIONE, B. (19’77) Arch. Biochem. Biophys. 182, 64. 4. LEVY, M., SIXENKEIN, I., AND FISHMAN, L.

(1969) in Methods in Enzymology (Lorand, L., and Perlman, G. E., eds.), Vol. 19, p. 672, Academic Press, New York. 5. TAYU)R, J. M., MITCHELL, W. M., AND COHEN, S. (1974) J. Biol. Chem. 249, 2133. 6. GRAY, W. R. (1967) in Methods in Enzymology

(Him, H. W., ed.), Vol. 11, New York. 7. HARTLEY, B. S. (1970) Biocltem. 8. LAI, C. Y. (1977) in Methods (Hirs, H. -W., ed.), Vol. 47, New York. 9. ZEEUWS,

R.,

AND

Academic Press, J. 119, 805. in Enzymology Academic Press,

STROSBERG,

A.

D.

(1978)

FEBS Lett. 85, 68. 10. DAYHOFF, M. E. (1976) Atlas of Protein Sequence and Structure, Vol. 5, Nat. Med. Res. Found., Georgetown Univ. Medical Center, Washington, D. C. 11. SCHALLER, E., AND VON DEIMLING, 0. (1979) Anal. Biochcm. 93, 251. 12. LETOW, LING, 13. GRESIK,

V.,

GROSZARTH,

C.,

AND

VON

DEIM-

0. (1979) Histochemistry E., AND BARKA,

60, 237. T. (1977) J. Histochem.

Cytochem. 25, 1027. 14. BARKA,

T., VAN DER NOEN, H., MICHELAKIS, A. M., AND SCHENKEIN, I. (1980) Lab. Invest. 42, 656.