New biological functions of intracellular proteases and their endogenous inhibitors as bioreactants

NEW BIOLOGICAL FUNCTIONS OF INTRACELLULAR PROTEASES AND THEIR ENDOGENOUS INHIBITORS AS BIOREACTANTS NOBUHIKO KATUNUMA The University of Tokushima, Institute for Enzyme Research, Tokushima 770, Japan

INTRODUCTION

An impressive body of knowledge has accumulated in the past 10 years in the area of the protein chemistry and enzyme mechanisms of proteases and their endogenous inhibitors. Recently, cell biological studies at the molecular level have been initiated in this line with the epoch-making developments of gene technology, cell engineering, X-ray crystallography and computer homology search. With this historical background I am especially interested in the evidence that some proteases and their endogenous inhibitors, located in the membrane of cells and organelles, participate in the functions of specific biological information and recognition. The relationship between intracellular proteases and their endogenous inhibitors is not only important for intracellular protein catabolism and its suppression, but also in the regulation of processing of biologically active proteins by limited proteolysis. Recently, it has been taken up as a key and key-hole relationship which plays an important role for special recognition apparatus of biological information, like the relationship between peptide hormones and their specific receptors. Many unexpected biological functions as bioreactants of the intracellular proteases and their endogenous inhibitors have been found recently, e.g., a kind of tryptase in the helper T-cell membrane is a recognition site of AIDS virus (HIV) and the precursor of Alzheimer's deposition protein ~ contains a Kunitz-type trypsin inhibitor domain. H-ras product p21 is a cathepsin inhibitor of the cystatin family.

P R E S E N T A D V A N C E S IN T R Y P T A S E R E S E A R C H

Structure and Function of Mast Cell Tryptase (Tryptase M) Two types of serine proteases have been identified in the mast cell family and basophile cells, named chymase type and tryptase type. Two types of chymotrypsin-like proteases have been first identified by us in rat mucosal 377

378

N. KATUNUMA

mast cells and connective tissue mast cells, and these have been named chymase (Mast Cell Serine Protease I, MCSP-1) and atypical chymase (MCSP-II), respectively (1, 2). The trypsin like serine protease in connective tissue mast cells, named tryptase, was also purified from human pulmonary mast cells (3, 4) and from rat peritoneal mast cells (1). These serine proteases were shown by immunohistochemical staining to be localized in histamine granules. During the past ten years, rat chymase, atypical chymase and tryptase have been purified and characterized by our group and the amino acid sequences of these have been determined (5-8). Both chymase and tryptase M in mast cell histamine granules play important roles in the process of IgE mediated degranulation and in the formation of allergic inflammation. The inhibition of chymase activity inhibits histamine release induced by IgE-receptor mediated but not that induced by Ca ionophore, but inhibition of tryptase does not show any inhibitory effect on histamine release. Therefore, chymase-type protease activity in histamine granules plays an essential role in IgE-receptor mediated degranulation. However, little is known about the biological roles of chymase and tryptase-type proteases after degranulation. Tryptase, a trypsin-type protease that cleaves peptide and ester bonds on the carboxyl side of basic amino acids, was purified from human pulmonary mast cell by Schwartz et al. (3) and also from peritoneal mast cells by Katunuma et al. (1, 2). These tryptases (tryptase M) were shown immunohistochemically to be localized in secretory granules of mast cells. The tryptase Ms from human and rats are tetrame~s, with apparent molecular weights of 144,000 and 142,000, respectively?~ Human tryptase is composed of two subunits of 37,000 and two subunits of 35,000 (3, 4), and the rat tryptase consisted of four identical subunits of 35,000, each subunit having one active site (1, 2). T As shown in Table 1, mast cell tryptase hydrolyzes a !specific substrate for factor Xa more rapidly than other synthetic substratgs of trypsin-type proteases tested. The apparent K m values of tryptase M and factor Xal for prothrombin were 2.3 p,M and 0.74/&M, respectively, and the KcatS were 46.3s -I and 10.3s -1. Therefore, the proteolytic efficiency (Keat/Km) Of tryptase M was 1.4-fold higher than that of factor Xa. By product analysis, the tryptase M converts prothrombin to thrombin without addition of Va-~hospholipid and Ca 2+ as cofactors (9). These results indicate that tryptase i M contributes to intravascular blood coagulation and also to deposit fibrifi in inflammatory tissues. Each subunit of tryptase M is bound with a spgcial endogenous inhibitor named trypstatin in the histamine granules as shown in Figure 1, and the tryptase M and trypstatin dissociate in the extracdlular milieu after IgE-receptor mediated degranulation. Therefore, trypstatin secreted from

379

FUNCTIONS OF INTRACELLULAR PROTEASES

,Q

In~" granule

After de,granulation

FIG. 1. Stereostructure of tryptase M and trypstatin.

Intrinsic pathway = Fibrin

Rbrinogen

t

Extrinsic pathway

Va - PL Ca z+ •

Prothrombin

a - Thrornbin Ca 2+ X III

Tn/ptase

Tqtpstatin

(Rat mast cells)

= X llla

Cross4inking Fibrin

FIG. 2. Mode of action of tryptase M and trypstatin on fibrin deposition.

"-

380

N. KATUNUMA TABLE 1. ACTIVITIES OF TRYPTASE AND CHYMASE ON SYNTHETIC SUBSTRATES Relative activity*(%)

Substrate (10 #M) Boc-Phe-Ser-Arg-MCA (Trypsin)# Boc-Val-Pro-Arg-MCA (a-Thrombin) Boc-Val-Leu-Lys-MCA (Plasmin) Boc-Ile-Glu-Arg-MCA (Fractor Xa) GIt-GIy-Arg-MCA (Urokinase) Pro-Phe-Arg-MCA (Kallikrein) Bz-Arg-MCA (Trypsin) Suc-Ala-Ala-Pro-Phe-MCA (Chymotrypsin) Suc-Leu-Leu-Val-Tyr-MCA (Chymotrypsin)

Tryptase trypstatin complex

Tryptase

100 80.3 4.7 4.1 3.5 0 1.2 0 0

100 50.6 6.1 149.0 4.6 4.6 0 2.0 12.3

Chymase 0 0 2.6 0 0 4.6 1.2 26.6 100

*Values are activities of tryptase as percentages of that with Boc-Phe-Ser-Arg-MCA and those of chymase as percentages of that with Suc-Leu-Leu-Val-Tyr-MCA. tThe enzymes for which the compounds are substrates are shown in parentheses.

mast cells around granules may act as an anticoagulant by inhibiting both intravascular and extravascular prothrombin activation. The functions of tryptase M and trypstatin are summarized in Figure 2.

Structure and Function of T-Lymphocyte Tryptase (10, 11) (Tryptase TL) We purified two kinds of tryptase M-like new proteases from the cultured human helper T-lymphocytes, Molt 4, clone 8. We named these tryptase TL-1 and tryptase TL-2. The purified tryptase TL-1 is homogeneous on agarose gel electrophoresis and has a molecular weight of 400kDa. The tryptase TL-1 is composed of 12 heterogeneous subunits which are distributed between 24K and 39K on two dimensional SDS-PAGE and isoelectric focussing. However, no immunocrossreactive subunits with anti-tryptase M antibody were observed. The tryptase TL-2 preparation is also homogeneous and had a molecular weight of 200kDa. The molecule is composed of two identical subunits of 38kDa plus four identical subunits of 31kDa. The tryptase TL-2 is immunocrossreactive with anti-tryptase M antibody and is localized in the membrane of Molt 4, clone 8 cells. The inhibition patterns of tryptase TL-1 and TL-2 by various protease inhibitors are summarized in Table 2. Tryptase TL-1 and TL-2 are inhibited very strongly with trypstatin and leupeptin, while very weakly with aprotinin (PSTI), BBI, and al-antitrypsin in the same manner as that of tryptase M.

381

FUNCTIONS OF INTRACELLULAR PROTEASES TABLE 2. INHIBITION PROFILE OF TRYPTASE TL-I AND TL-2 BY VARIOUS INHIBITORS Residual activity (%)

None

Trypstatin (10 ~M) Trypstatin (1 t~M) Leupeptin a2-Macroglobulin (100 ~g/ml) PSTI Antipine HIATI (0.1 t~M) a~-Antitrypsin TPCK BBI Chymostatin Pepstatin E-64 c

Tryptase T~

Tryptase T2

100 19 53 21 43 56 56 70 71 71 79 88 100 74

100 8 13 56 4 63 90 98 80 89

If no number is indicated, 10/,~Mwas added.

STRUCTURES

AND INHIBITORY CHARACTERISTICS ENDOGENOUS TRYPSIN INHIBITORS

OF NEW

Natural trypsin inhibitors are widely distributed in microorganisms, plants and animals and have been well characterized. Among them, Kunitz-type trypsin inhibitors are representative. We will introduce the structures and functions of new Kunitz-type trypsin inhibitors, such as trypstatin and A4-inhibitor in amyloid protein precursor from neuritic plaques of patients with Alzheimer's disease.

Structures and Properties of Trypstatin (4, 12) We found a new endogenous trypsin inhibitor that associates with tryptase M in histamine granules of rat mast cells. The inhibitor, named trypstatin, was purified and the amino acid sequence was determined and found to be a new Kunitz-type inhibitor. The sequence of trypstatin is compared with those of other known Kunitz-type protease inhibitors in Figure 3. The reactive site of trypstatin which is a highly conserved region of this Kunitz-type inhibitor may be the -G-P-C-R-A- domain. Trypstatin inhibited the activities of trypsin and tryptase M by forming a complex in a molar ratio of 1 : 1 with trypsin and with one subunit of tryptase, respectively. It caused the greatest inhibition of rat mast cell tryptase (K i = 3.6 x 10-10 M) and factor Xa (K i = 1.2 x 10-10 M). The inhibition of mast cell chymase (K i = 2.4 x 10-8 M) and pancreatic trypsin (K i = 1.4 x 10-8 M) were

382

N. KATUNUMA Homology with trypstatin

(1) (2) (3) (4) (5) (1) A4 - 751 amiloid protein (from neuritic plaques of "} (Alzheimer's disease) I Homology 36% (2) Trypstatin (rat mast cells) [Katunuma at el.] (3) Inter-a-trypsin inhibitor (Bovine) (4) Inter-a-trypsin inhibitor (human) (5) Aprotinin (bovine) FIG. 3. Homology of trypstatin with various Kunitz-type trypsin inhibitors.

observed also as shown in Table 3. The trypstatin also strongly inhibits tryptase TL-1 and TL-2. The homology search indicated 70% sequence identity of its inhibitory domain with those of inter-a-trypsin inhibitors. The identity with aprotinin is not so high (35%). Since human inter-a-trypsin inhibitor is identical to an endothelial cell growth factor 2b from human hepatoma, the trypstatin may have a function as a growth stimulative factor for endothelial cells.

Properties of A4 Inhibitor in Alzheimer Amyloid Precursor Protein The total amino acid sequences of the precursor of amyloid protein 13 (APP) in senile plaque with Alzheimer disease are deduced from nucleotide sequences of the cDNA by Grenner (13), Masters (14), Tanzi (15), Ponte (16) and Kitaguchi (17), recently. It has been established that there are at least three types of APP mRNA in various organs, which encode APP-695, APP-751 and APP-770. Gene expression of Alzheimer precursor protein is illustrated in Figure 4. APP-695, APP-751 and APP-770 are expressed by a combination of four exons, H I, J and K. The APP-751 and APP-777 have 56 and 57 amino acids inserts at position 288 from the N-terminus of APP-695. The inserts of APP-751 and APP-770 contain identical 56 amino acid sequences which shows a typical structure of a Kunitz-type trypsin

FUNCTIONS OF INTRACELLULAR PROTEASES

383

TABLE 3. INHIBITORY EFFECTS OF TRYPSTATIN ON VARIOUS PROTEASES Protease

Substrate

pH

Value of Ki (M)

Pancreatic trypsin Mast cell tryptase Factor Xa a-Thrombin Chymase Pancreatic elastase Papain

Boc-Phe-Ser-Arg-MCA Boc-Phe-Ser-Arg-MCA Boc-IIe-GIu-Gly-Arg-MCA Boc-Val-Pro-Arg-MCA Suc-Leu-Leu-Val-Tyr-MCA Suc-AIa-Pro-AIa-MCA Z-Phe-Arg-MCA

7.5 8.5 8.0 8.0 8.0 7.0 6.0

1.4 × 10-8 3.6 x 10-1° 1.2 x 10-l° No inhibition* 2.4 × 10-s No inhibition* No inhibition*

*No inhibition at the concentration of 1 /~Mof trypstatin.

i n h i b i t o r . W e s t u d i e d t h e i n h i b i t o r y profile o f t h e A 4 - i n h i b i t o r d o m a i n using the recombinant total inhibitor domain. The A4-inhibitor strongly i n h i b i t e d p l a s m i n a n d t r y p t a s e M activities at K i v a l u e s o f 10 -11 M a n d 10 -10 M, r e s p e c t i v e l y , a n d also i n h i b i t e d t r y p s i n a n d c h y m o t r y p s i n at t h e 10 -9 M levels o f t h e i r K i v a l u e s as s h o w n in T a b l e 4 (10, 11). H o w e v e r , thrombin, factor Xa, chymase, elastase and urokinase are not inhibited.

Expression of alzheimer precursor proteins

APP- 751 -

(APP - 751 structure)

Membrane

~

::::)::::=:ii::::i ii i gi)iiiii::ii i ::if::ii::i C~i iiiiiiiiiiiiiiiii!ii~iiiill A4 - inhibitor NH2

0!

t 300

t Eoo

COOH

~ - amyloid t 700

Amino acid residues

FIG. 4. Gene expression of precursor proteins of Alzheimer amyloid.

COOH

384

N. KATUNUMA TABLE 4. Ki VALUES OF A4-INHIBITORFOR VARIOUS PROTEASES

Protease

Substrate

pH

Ki value nM

Trypsin Tryptase M Factor Xa a-Thrombin Chymase a-Chymotrypsin Elastase Plasmin Urokinase Plasma k a l l i k r e i n Tissue kallikrein Papain Cathepsin B

Boc-Phe-Ser-Arg-MCA Boc-Phe-Ser-Arg-MCA Boc-lle-Glu-Gly-Arg-MCA Boc-VaI-Pro-Arg-MCA Suc-Leu-Leu-Val-Tyr-MCA Suc-Leu-Leu-VaI-Tyr-MCA Suc-AIa-Pro-AIa-MCA Boc-Val-Leu-Lys-MCA GIt-GIy-Arg-MCA Z-Phe-Arg-MCA Pro-Phe-Arg-MCA Z-Phe-Arg-MCA Z-Phe-Arg-MCA

7.5 8.5 8.0 8.0 8.0 8.0 7.0 7.5 7.5 7.8 7.8 6.0 6.0

2.7 0.22 257.0 NI* NI 8.5 NI 0.075 NI 73.9 28.4 NI NI

*NI, no inhibition at a concentration of 1 /~M A4 inhibitor, Suc-, succinyl-; Z-, benzyloxycarbonyl-.

NEW B I O L O G I C A L FUNCTIONS AND P A T H O L O G I C A L S I G N I F I C A N C E OF E N D O G E N O U S P R O T E A S E I N H I B I T O R S

Receptor of HIV-1 and Tryptase-Trypstatin System in Helper T-Cells (12, 18) The A I D S virus ( H I V - l ) , a causative agent of acquired immunodeficiency syndrome, infects cells via interactions between cellular receptors (CD4) and the external envelope glycoprotein (gpl20). However, the mechanism by which HIV-1 enters host cells after binding has not been clarified. The post binding events of HIV-1 on cell surfaces are necessary for infection of cells by HIV-1. Experiments using synthetic peptides, encoding gpl20, and a neutralizing monoclonal antibody indicate that the epitope consisting of 24 amino acids at positions 308-331 of gpl20 (NNT24) is essential for infection by the HIV-1 (19). However, the epitope portion of gpl20 is different from the domain of gpl20 at positions 367-439, which plays a role in the binding of gpl20 to CD4 molecules. These findings suggest that the neutralizing epitope might be accessible to an unknown cell membrane receptor upon infection. As mentioned earlier in the text, a new cellular Kunitz-type protease inhibitor, trypstatin, has been purified and the amino acid sequences have been determined. Coincidentally, six of the amino acid sequences in the active site of trypstatin are identical with the neutralizing epitope 13 of gpl20 (NNT24) of HIV-1 as illustrated in Figure 5. Since trypstatin inhibits

FUNCTIONS OF INTRACELLULARPROTEASES

385

tryptase activity specifically, we have examined the effects of trypstatin and anti-tryptase antibody on HIV-1 infection using the HIV-1 susceptible T-ceU line, Molt 4, clone 8. The effects of trypstatin and anti-tryptase M antibody on syncytium formation caused by Molt 4, clone 8 cells are shown in Figure 6. The formation of syncytia by HIV-1 infection was inhibited completely by trypstatin at a concentration of i /xM and this inhibition was also observed at a lower trypstatin concentration of 300 nM (c and d). Also, anti-tryptase M antibody at a concentration of 600/xg/ml inhibited the syncytium formation (e and f). Cellular tryptase M antigen densities on the two types of cell lines, susceptible and nonsusceptible cells, were compared using cell surface staining with anti-tryptase M antibody in Table 5. Therefore, the relation of tryptase M with trypstatin and the recognition site of epitope 13NNT24 of HIV-1 with the receptor of helper T-cells are the common keys (Fig. 8). These findings suggest that the tryptase M-like protease located in Molt 4, clone 8 membranes might be involved in HIV-1 infection as a recognition site of epitope 13 of HIV-1. As mentioned before in this article, we found tryptase M-like new proteases from membranes of cultured Molt 4, clone 8 cells, named tryptase TL-1 and tryptase LT-2. The tryptase TL-2 is inhibited strongly by trypstatin and is crossreactive with anti-tryptase M antibody. It is important to note that the tryptase TL-2 cuts the NNT24 of gp120 epitope 13 between G and R in a limited proteolytic manner as shown in Figure 7. This evidence suggests that the tryptase LT-2 plays the most important role as the recognition receptor of HIV epitope 13 in the infection of HIV to helper T-cells. Possible role of tryptase TL-2 in Molt 4, clone 8 in the HIV-infection is illustrated in Fig. 9.

Kunitz-type A4-inhibitor in the Precursor Protein of Alzheimer Amyloid The pathogenesis of Alzheimer disease and Alzheimer type senile dementia is characterized by the atrophy of specific neuronal populations and angiopathy due to the intracellular accumulation of cytoskeletal constituents, paired helical filaments (PHF) and tau-protein, and by the extracellular deposition of highly insoluble protein, named amyloid protein 13or A4-protein. The APP-751 and APP-770 contain a Kunitz-type trypsin inhibitor domain (exon I). Further evidence supports the notion that the special proteases and/or their inhibitors may be critically involved in the deposition of amyloid proteins to manifest the Alzheimer pathogenesis. The mRNA blot analysis of APPs by Kitaguchi et al. (17, 20) showed no changes in the expression of APP-695 and APP-751 mRNA in the frontal cortex of Alzheimer patients, compared with that of the age-matched control, while APP-770 mRNA level was significantly elevated in Alzheimer patients.

386

N. KATUNUMA

(Trypatatin) I A A C N L P ( ~ ) V

O (~)(~)C ( ~ ) ' ~ ) ( ~ ) A E L L

C

(Kido et al. 1988)

316

R

R

~

V

~NN i z41

30e , ~ q ~

_R M

PR (gp 120) N m

T VQ L NtQ S V E I N~C ~ 295

I

303

AtJ H C N'I S R A K W N N ' ~ C 341

350

(Goudsmit et al. 1988)

* Potential Asn- linked glycosylation site FIG. 5. Amino acid alignments of the neutralizing epitope 13 of gpl20 of HIV-1 and trypstatin.

In other words, the inhibitor domain increases in the brain of Alzheimer patients. As shown in Table 4 on the inhibitory profile of A4-inhibitor, the inhibitory characteristics are different from that of trypstatin. We made very strong antibody against the A4-inhibitor domain and detected immunohistochemically the cellular localization of precursor protein having A4-inhibitor (APP-751 and APP-770) in various organs of normal rats

TABLE 5. COMPARISON OF CELL SURFACE STAINING OF ANTIGEN POSITIVE CELLS WITH ANTITRYPTASE M ANTIBODY Cells

(%)

Molt-4-clone 8 Molt-4 S KT- 1B resting T* activated T

96.6 87.0 7.9 0.2 7.8

*Indicates CD3-positive cells in nonactivated PBMC.