Proteolytic Inactivation of CY- 1-Anti-chymotrypsin - The Journal of ...

OF BIOLOGICAL CHEMISTRY THE JOURNAL

Vol. 266,No. 32, Issue of November 15, pp. 21482-21487,1991 Printed in U.S.A.

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Proteolytic Inactivationof CY- 1-Anti-chymotrypsin SITES OF CLEAVAGEAND GENERATION OF CHEMOTACTIC ACTIVITY* (Received for publication, May 17,1991)

Jan Potempa$, Danuta FedakS, Adam Dubin$, Alan Mast%, and James TravisllII From the $Institute of Molecular Biology, Jagiellonian University, 31-120 Cracow, Poland, the $Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, and the 9Department of Biochemistry, University of Georgia, Athens, Georgia 30602

The effect of several microbial and mammalian pro- states which correlate with a genetic deficiency in specific teinases on the inhibitoryactivity of human plasmaa- serpins have previously been described, the best example 1-anti-chymotrypsin(a-1-Achy) has been tested. Most being that of a-1-PI where a deficiency state has been correof these enzymes caused rapid inactivation of the in- lated with the development of pulmonary emphysema (3). hibitor by cleavage at single sites within thereactiveOne of the more interesting serpinsis a-l-anti-chymotrypsite loop between Pa Lys and P3’Leu, with additional sin (a-1-Achy), an importantacute phase plasma protein cleavages also being detected in some cases near the whose concentration canrise up to$fold during inflammatory NH, terminus of the native protein. In contrast,two of episodes (4,5). The regulatory function of thisinhibitor the enzymes tested failed to inactivate a-1-Achy, al- probably involves the control of neutrophil cathepsin G (cat though they could cause removal of peptides near the NH, terminus. Studies of neutrophil chemotaxis re- G) (6),although it can also inhibit human pancreatic elastase vealed that native or NH,-terminally truncated a-1- I1 (7) and mast cell chymase (8).An inherited, partial, heterAchy was not stimulatory. However, testing of two ozygous a-1-Achy deficiency state for this serpin has been enzymatically inactivated formsof the inhibitor (a-1- described (9) which has been associated with liver and lung Achy’), cleaved at widely different positions within the function abnormalities. However, no homozygotes have yet reactive-site loop, indicated that they had become po- been identified suggesting that such a defect might be lethal. More recently, a role inthe development of Alzheimer’s tent chemoattractants at concentrations within the nanomolar range. Competition studies using proteolyti- disease has been suggested because of the presence of this inhibitor in the brain amyloid plaque deposits (10). cally inactivated a-1-proteinase inhibitorsuggested Kress and co-workers (11-14) have previously shown that that the chemotactic activity of the two inactivated serpins was probably mediated by the same mechaa-1-Achy is more sensitive to proteolytic inactivation by nism. The physiological relevance ofthis chemotactic microbial and snake venom proteinases than are other seractivity is underscored by the results of plasma elimi- pins. In these studies inactivation of the inhibitor by a pronation studies which indicate thata-1-Achy’ is elimi- teolytic cleavage within the inhibitor reactive-site loop was nated at approximately the same rate as native a-1- suggested but no structural data were presented for confirAchy, thus prolonging chemotactic stimuliwithin the mation. In thisreport we describe the kinetics of inactivation tissues. of a-1-Achy by bacterial proteinases, porcine andhuman trypsins, human plasmin, andhuman neutrophil elastase (HNE), aswell as their cleavage sites within the reactive-site loop of this inhibitor. To assess the physiological importance Proteinase inhibitors represent approximately 10% of the proteins in human plasma, suggesting that they have major of these proteolytic cleavages we have also determined the roles in regulating proteolytic activity within the body. Most plasma elimination time of the modified forms of a-1-Achy. of these inhibitors arespecific for serine proteinases, belong- Finally, we have followed the generation of neutrophil cheing to a superfamily of structurally related proteins referred motactic activity as native a-1-Achy is converted proteolytito as serpins (1).Their function is to regulate a variety of cally into inactive forms. Since it is confusing to describe the different forms of a-lproteolytic events, including those used in phagocytosis (a-lAchy generated by the proteinases used here, the following proteinase inhibitor (a-1-PI)’),coagulation (antithrombin 111, heparin cofactor 11, and protein C inhibitor), fibrinolysis (a- nomenclature will be used. Native a-1-Achy will refer to the fully active inhibitor. a-1-Achy’ will refer to the form of the 2-antiplasmin and plasminogen activatorinhibitors),and complement activation (Cl inhibitor) (2). Different disease inhibitor inactivated by a proteolytic cleavage within the reactive-site region. a-1-Achy which is cleaved at the amino * This work was supported in part by grants from the National terminus will be referred to specifically. Heart, Lung and Blood Institute andby Grants CPBP04.01.2.10 and 04.02.1.3.1 from the Polish Academy of Sciences and the Polish EXPERIMENTALPROCEDURES Ministry of Higher Education, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. Materials This article must therefore be hereby marked “advertisement” in All synthetic peptide proteinase substrates used in this study, p accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nitrophenyl-p’-guanidinobenzoate, o-phenanthroline, diisopropyl 11 To whom correspondence should be addressed. The abbreviations used are: a-1-PI, a-1-proteinase inhibitor; cat fluorophosphate, and iodoacetamide, as well as the enzymes porcine G, cathepsin G; a-1-Achy, a-1-anti-chymotrypsin; HNE, human neu- trypsin, thermolysin, and clostripain were from Sigma. Stuphylococtrophil elastase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide cus aurew metalloproteinase and V8 proteinase (151, HNE and cat G (16), and human cationic trypsin (17), were isolated as previously gel elctrophoresis; FMLP, N-formyl-L-methionyl-L-leucyl-L-phenyldescribed. Pseudomonas aeruginosa elastase and Serratia marcescens alanine.

’

2 1482

Anti-chymotrypsin Chemotactic Activity

21483

attractants placed in the lower compartment by a cellulose nitrate micropore filter with a mean pore diameter of 5 pm (Sartorius). After 1h the chambers were disassembled and themembranes were stained with hematoxylin. Chemotaxis was determined as themean depth in micrometers of penetration into filter of the leading two cells.Experiments were performed in triplicate. In competitive experiments neutrophils were incubated in 10 nMof either native a-1-PI, a-lAchy, or proteolytically inactivated forms of each inhibitor for 30 Methods min at ambient temperature, then washed three times with medium. Assay of Enzyme Activity-Trypsin amidase activity was measured The cells were then assayed for a chemotactic response to 1 nM a-lwith Bz-L-Arg-pNA (final concentration 1.0 mM) in 0.2 M Tris-HC1, PI', 1 nM a-1-Achy' or 10 nM N-formyl-L-methionyl-L-leucyl-L2 mM CaC12,pH 8.0. HNE and catG activities were measured in 0.1 phenylalanine (FMLP). M Tris-HC1, 0.5 M NaC1, pH 7.4, using Suc-L-Ala-L-Ala-L-Ala-pNA and Suc-L-Ala-L-Ala-L-Pro-L-Phe-pNA (finalconcentrations 0.5 RESULTS mM), respectively. The reactions were monitored by following the Enzymatic Inactivation of a-1-Achy-Incubation of a-lchange in Alas at 25 'C. The proteolytic activities of bacterial metalloproteinases and porcine trypsin were measured with hide powder Achy with each of the threemicrobial metalloproteinases, the azure (Calbiochem) ( h a 1 substrate concentration 1%w/v) in 0.1 M two pancreatic trypsins, or HNE resulted in a concentrationTris-HC1, 2 mM CaCI,, pH 7.5. and time-dependentdecrease in itsinhibitory activity toward Active-site Titrations-Measurement of the activities of human cat G (Fig. 1).On an individual basis, a-1-Achy was inactiand porcine trypsins, as well as human plasmin, were performed with p-nitrophenyl-p'-guanidinobenzoate(20). Active-site titrated porcine vated most efficiently by P. aeruginosa elastase, followed by trypsin was also used to determine the inhibitory activity of a-1-PI S. mrcescem 56-kDa metalloproteinase, human pancreatic and a-2-macroglobulin. Theseinhibitors were used as secondary trypsin, and S. aureus metalloproteinase. HNE and porcine standards to determine the activities of HNE andcat G (a-1-PI) and pancreatic trypsin were less effective, causing inactivation of bacterial metalloproteinases (a-2-macroglobulin). Proteolytic Inactivation of a-1-Achy-Constant amounts of inhib- a-1-Achy at inhibitor:enzyme molar ratios 1 order of magniitor (final concentration 7.5 M) were incubated with individual pro- tude higher. On the other hand,a-1-Achy was totally resistant teinases in 0.05 M Tris-HC1, 1 mM CaC12,pH 7.4, at 37 "C in a final to inactivation by either clostripain or the 5'. aureus V8 volume of200pl. The molar ratio of a-1-Achy to proteinase was proteinase, which cleaved only at the amino terminus (Table adjusted so that proteolytic inactivation could be followed during a I), and only slowly inactivated by human plasmin (at a molar 10-120-min incubation period. Controls consisted of inhibitor and buffer only. At giventime intervals aliquotswere removedand tested ratio of 51, 40% of the inhibitory activity was retained after for inhibitory activity toward cat G by addition of an excess of enzyme. 2 h) (data not shown). In all cases inactivation was accomAfter a 5-min preincubation at 25 "C, the mixture was assayed for panied by limited proteolysis, as demonstratedby the appearresidual cat G amidolytic activity as described above. The effective- ance of a small peptide of M,near 5,000 (Fig. !?A). Despite ness of inhibitor cleavage was confirmed by SDS-polyacrylamide gel the removal of this peptide an increase in the rateof migration electrophoresis (21) using a 7-20% acrylamide (w/v) linear gradient. For chemotaxis and plasma elimination studies, proteolytically of the larger a-1-Achy fragment in SDS-PAGE was only cleaved serpins were separated from proteinases by Mono Q fast observed in the case of the trypsin-treated inhibitor (Fig. 2B, lane 2). This same pattern of a-1-Achy versus a-1-Achy' protein liquid chromatography. Sequence Anulysis of Modified a-1-Achy-a-1-Achy was incubated migration in SDS-PAGE has been described previously (25). with a given proteinase for a suitable time period in molar ratios Coincidentally, AT 111, in its proteolytically cleaved form, has ranging from 1:l to 3000:1, as described above, until residual cat G inhibitory activity had decreased to about 10%. Proteinase activity a mass which is 5 kDa lower than the native protein, yet was then inhibited by addition of either 1.0 mM diisopropyl fluoro- migrates more slowlyin SDS-PAGE than thenative inhibitor phosphate or 20 mM o-phenanthroline, depending on the enzyme (25). At the present stage we can only speculate that a change being tested, and the digestion mixture was either used directly for in protein tertiary structure is responsible for this unusual NHz-terminal sequence analysis or subjected to Mono Q fast protein liquid chromatography in 20 mM Tris-HC1, pH 8.0 to separate the inactivated proteinase from cleaved inhibitor. The latter was then lyophilized and desalted on Sephadex G-25. NHn-terminalsequence analysis was performed on eitherthe digestion mixture or the purified a-1-Achy' using an Applied Biosystem 470A gas-phase sequenator and the program designed by the manufacturer. In the case of a-lAchy' obtained from HNE digests, small peptide fragments were obtained by reverse-phase high performance liquid chromatography using an acetonitrile/trifluoroaceticacid gradient as described previously in analyzing the cleavage products of P. aeruginosa elastaseinactivated a-1-PI (22). Peptides were detected by monitoring at 214 nm, the individual fragments collected and dried under vacuum, and sequence analysis performed. Plasm Elimination St~dies-'~~I-labeled proteins (1.0 pg), radioiodinated by the lactoperoxidase/glucose oxidase method (Bio-Rad) were injected into the lateral tail vein of CD-1 mice. Blood samples (25 11) were obtained at timed intervals by retroorbital puncture and 1251measured in a y-counter. The initial time point,taken between 5 W and 10 s after injection, was considered to represent 100%radioactiv(L MINUTES OF REACTION ity in the circulation. Each labeled protein was tested at least in duplicate. FIG. 1. Time- and concentration-dependent inactivation of Chemotaxis-Chemotactic activity was determined in modified a-1-Achy by mammalian and bacterial proteinases. Inhibitor Boyden chambers by the micropore-filter leading front assay (23), was mixed with HNE (A), human pancreatic trypsin ( B ) , porcine using neutrophils obtained from peripheral blood of healthy donors pancreatic trypsin (C), S. aureus metalloproteinase (D), S. marcesand separated on Ficoll-Hypaque density gradients (Mono-Poly re- cem 56-kDa metalloproteinase (E), or P. aeruginosa elastase ( F ) at solving medium, Flow Laboratories) (24). Briefly, 1.2 X lo6 neutro- the indicated molar ratios as described under "Experimental Procephils/ml in RPMI medium 1640 (GIBCO) were placed in the upper dures." Aliquot8 were taken at given time periods and assayed for compartment of the Boyden chamber and separated from chemo- residual cat G inhibitory activity. 56-kDa metalloproteinase were kind gifts of Dr. J. Powers (Georgia Institute of Technology, Atlanta, GA) and Dr. A. Kreger (Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC), respectively. Human a-1-Achy, a-1-PI, a-2-macroglobulin, and plasminogen were purified from one batch of plasma by sequential affinity chromatography (18). Human plasmin was prepared by activation of plasminogen (19).

21484

ActivityChemotactic Anti-chymotrypsin TABLEI Cleavage sites on a-1-anti-chymotrypsin for bacterial and mammalian proteinases

1 2 3 4 5 1312 14 15 16 17 18 19 20 N-terminal of a-1-Achy (Residues 1 - 20): His Pro Asn Ser Pro - - - - - Thr Gln Glu Asn Gln Asp Arg Gly Thr B A

Pg P7 P6 P5 P4 P3 P2 PI' P3' P4' P5' P6' Reactive Site Loop of a-1-Achy (Residues 351- 364): Thr Ala Val Lys Ile Thr Leu Leu Ser Ala Leu Val Glu Thr C D E F

Cleavage sites not affecting a-1-Achy inhibitoryactivity: Site A- S.aureus VO proteinase Site B - human or porcine trypsin and clostripain

Inhibitorinactivatingcleavages:

Site C - human and porcine pancreatic trypsins Site D - human neutrophil elastase Site E- S.marcescens 56K metalloproteinase Site F - S.aureus metalloproteinase and P.aeruginosa elastase

Amino acidsequence

found in samples of a-1-Achy incubated withdifferentproteinases.

S.marcescens 5 6 K metalloproteinase

HIS ALA

PRO LEU

ASN VAL

SER GLU

PRO THR

LEU ARG

ASP THR

GLU GLU ILE VAL

S.aureusmetalloproteinseandP.aeruginosaelastaseHISPROASNSERPROLEUASPGLUGLUASN LEUVALGLUTHRARGTHRILEVALARG Humanneutrophilelastase

HISPROASNSERPROLEU ARGTHRVALALALEULEUTHR

PHE ASPGLU

Trypsins

GLY ILE

Clostripain

GLYTHR

HISVALASPLEUGLYLEUALASER

ASN

ASPARG

S.aureus

V8

proteinase

difference in the electrophoretic mobility between native and cleaved serpins. SequenceAnalysis of Enzymatically Inactivated Forms of a1-Achy-Amino-terminal sequence analysis of a-1-Achy after treatment with each of the threebacterial metalloproteinases gave identical results (TableI), yielding 2 residues/cycle. One residue in each cycle corresponded to the amino terminus of the native inhibitor. This was 2 residues longer than originally reported (26), beginning with the sequence NHn-His-Pro-. However, this result is consistent with the amino terminus predicted from the cDNA sequence reported earlier (27). It is also in agreement with the sequence of an a-1-Achy isoform recently described by Lindmark et al. (28). The second sequence represented the amino terminus of the new peptide generated by proteolytic cleavage within the reactive site of a-l-Achy. a-1-Achy which was treated with either human or porcine trypsin also gave 2 residues/cycle. One sequence was due to proteolysis between Arg-18 and Gly-19 and is apparently responsible for the faster migration of trypsin-cleaved a-lAchy in SDS-polyacrylamide gel electrophoresis (Fig. 2), since metalloproteinase-modified a-1-Achy, in whichcleavage is specifically within the reactive-site loop, migrates at thesame rateasthe native inhibitor. However, the second set of residues identified represents the sequence occurring after a cleavage site within the reactive-site loop (Table I). Significantly, clostripain could only cleave after Arg-18, since there were no other Arg residues accessible for proteolysis by this enzyme within the reactive-site loop. Treatment of a-1-Achy with HNE yielded two major se-

THR

GLN

HISVALASP LEU LEU SER ALA LEU VAL GLU

ASN ARG

LEUGLYLEU

GLUASN ALA

SER THR

GLYTHRHISVALASPLEU

quences per cycle. One of the major sequences again represented the amino terminus of native a-1-Achy. The other major sequence was found to occur within the reactive-site loop and was confirmed by isolation and structural analysis of individual peptide fragments, as described under "Methods." In summary, the proteinases examined cleaved a-1-Achy within the reactive-site region between the P6 Lys and the PB'Leu, or within the first 19 residues of the amino terminus of the native protein. Significantly, after proteolytic cleavage in these regions there was no further degradation of the various forms of a-1-Achy', even after prolonged incubation at relatively high enzyme concentrations (Fig. 2B, lane 7). Plasma Elimination Studies-Cleavage of a-1-Achy within the reactive-site loop with HNE, near the amino terminus with clostripain, or at both siteswith trypsin had no effect on the rate of clearance of such modified forms of the inhibitor from the circulation, relative to that of the native protein (Fig. 3). Such results were obtained despite the fact that such cleavage causes major conformational changes in the structure of the inhibitor (29), similar to that reported earlier for a-lPI (30). The site within the reactive-site loop where cleavage occurs is also apparently not significant since a form of a-lAchy' obtained by cleavage between the P?' Arg and the Pa' Thr by a trypsin-like proteinase from Porphyromonus gingivalis was removed at the same rate as the HNE and trypsin cleaved protein.' This slow removal of various forms of a-lAchy' is in sharp contrast to the rapid, receptor-mediated plasma elimination of the a-1-Achy-cat Gcomplex, which is shown for comparison (Fig. 3). The slow elimination of a - l J. Potempa and A. Mast, unpublished observation.

Activity Chemotactic Anti-chymotrypsin

FIG.2. SDS-polyacrylamide gel electrophoresis of different a-1-Achy/proteinase reaction mixtures. Enzymes and inhibitor were incubated as described under “Experimental Procedures” and aliquots removed at specific times for analysis by gel electrophoresis after treatmentwith 1%SDS.A, a-1-Achy with P. ueruginosa elastase (3,0001inhibitor:enzyme molar ratio). Lune 1,inhibitor control; lanes 2-5, 20-,40-, 60-, and 240-min incubations, respectively; lane 6, standard proteins. B, a-1-Achy with human trypsin (lane 2 ) , HNE (lane3 ) ,S. aureus metalloproteinase (lane4 ) , S. marcescens 56-kDa metalloproteinase (lane 5), and P. ueruginosa elastase (lanes 6 and 7). Incubation times were for 60 min at 1001inhibitor:enzyme molar ratios (lane 7, 180 min). Lune 1, inhibitor control; lane 8, standard proteins.

21485 Achy‘ indicates that it is not recognized by the receptormediated pathways, which rapidly remove serpin-proteinase complexes fromthe circulation (31-33), further increasing the potential for their accumulation in the acute phase state. Neutrophil Chemotactic Activity of Various Forms of a - l Achy-The extraordinary sensitivity of a-1-Achy to conversion to a-1-Achy’ by proteolytic inactivation within the reactive-site loop, together with the slow clearance of each from the circulation, suggeststhat large amounts of a-1-Achy’ could accumulate ininfected andlor inflamed tissues. in vivo. Since this inactivation results ina major rearrangement in protein structure we investigated whether it might also lead to the generation of chemotactic activity, as previously described for various forms of a-1-PI’ (34, 35). In these experiments two forms of a-1-Achy‘ obtained by inactivation of the native protein with either trypsin or the S. aurew metalloproteinase were used becausethese proteinasescleave at widely separated regions within the reactive-site loop (Table I). In addition, trypsin also cleaves a-1-Achynear the amino terminus, yielding aform of a-1-Achy’which has been modified in two places. As shown in Fig. 4, both modified forms had potent neutrophilchemotactic activity.Significantly, neutrophils preincubated with either form of modified a-1-Achy or with P. aeruginosa treated a-1-PI did not migrate toward these sameproteinsin Boyden chamber experiments. However, similar pretreatment had noeffect on the neutrophil chemotactic response to FMLP (Fig. 5), indicating that this latter compound was beingrecognized by a separate receptor on the neutrophil surface. Controls using either native a-1-Achy or a-1-PI had no chemotacticactivity, andpreincubation of neutrophils with these proteins had no effect in reducing cell migration stimulated by the proteolytically modified inhibitors (Figs. 4 and 5). DISCUSSION

Members of the serpin family which have retained their inhibitory activity are all characterized by the presence of an exposed reactive-site loop that is sensitive to proteolysis by enzymes with which it cannot readily form stable complexes 100

t

* O t I

!’I ‘5 20 73

K

101 I 0

I

I

10

20

I

I

30 40 Time (rnin)

I

I

50

60

FIG.3. Comparison of plasma clearance of native versus various proteolytically cleaved forms of a-1-Achy. Native ininhibitor obtained from digest with: trypsin ,).( S. aureus hibitor (0); metalloproteinase (O), clostripain (A), cat G.a-1-Achy complex (W).

I

I

I

I

II

FIG.4. Chemotactic activity of a-1-Achy’ from S. aureus metalloproteinase and porcine pancreatic trypsin incubation mixtures. a-1-Achy’ wasobtained by incubation of native inhibitor with each enzyme as described under “Experimental Procedures.” Appropriate molar dilutions of each form of a-1-Achy’, as well as native inhibitor, were then assayed for their ability to stimulate chemotaxis of human neutrophils in modified Boydenchambers. The points represent the average of the triplicate determinations, and the error bars represent the standard error of the mean. Migration stimulated by 10 nM FMLP (the positive control) is represented by a striped bar. Spontaneous migration was26 f 3 pm. 0, S. aureus metalloproteinase-derived a-1-Achy’; 0, porcine pancreatic trypsinderived a-1-Achy’;A, native a-1-Achy.

21486 100

Activity Chemotactic Anti-chymotrypsin

1

Desensitizatlon: a,Achy’ (10’M)

n,PI’ (108M)

0 natlve a,Achy rnedla only

10’M

108M

~ o . ~ M

CHEMOATTRACTANT

FIG. 5. Human neutrophil chemotaxis before and after exposure to competitive ligands. Neutrophils were incubated for 30 min at room temperature with 10 nM each of a-1-Achy*(light striped burs), a-l-PI*(dark striped bars), native a-1-Achy (stippled bars), assay medium control (solid burs), as described under “Experimental Procedures,” and assayed for a chemotactic response to 1 nM of either or-1-Achy’ or a-l-PI*,or 10 nM FMLP. The datarepresent an average of triplicate determinations, and error burs represent the standard error of the mean. Spontaneous migration was 25 -t 2 Nm.

(11-14, 36). The crystallographic model of cleaved forms of serpins (29, 30) reveals that proteolysis within the loop is accompanied by a major change in protein tertiary structure and the transition from a “stressed” (S) structure to a more ordered, thermodynamically stable “relaxed” (R) form of the molecule (37). This conformational change appears to occur in other serpins with inhibitory activity (38, 39) and may provide a physiological switch that regulates inhibitory activity at inflammatory foci (37). However, from the data described above it is likely that theinactive forms of inhibitors have alternate functions which are of far more importance during acute conditions. Previously, native human a-1-Achy had been shown to have at least three functions, all of which appeared to involve the regulation of chymotrypsin-like proteinases,including cat G, mast cell chymase, and lymphocyte cell surface proteinases (2, 8, 40, 41). Cathepsin G can not only degrade connective tissue components (42, 43), it can also act as an effective angiotensin-converting enzyme (44) and an inactivator of bradykinin (45). Therefore, by regulating this activity a-lAchy may control both connective tissue breakdown and/or smooth muscle contraction at an inflammatory locus, although this has notyet been proven. In a different sense a-lAchy may also act as an immunoregulator, since incubation of the native inhibitor with killer lymphocytes leads to the loss of both killing and antibody-dependent cytolytic activity (40, 41). Recently, a function for HNE-inactivated a-1-Achy in the production of IL-6 and, indirectly, acute phase protein synthesis was demonstrated (46). We now have data which indicate an additional role for a-1-Achy’ in neutrophil chemotaxis, the results obtained indicatingthat thisactivity can be rapidly generated when the native inhibitor is cleaved by a wide array of bacterial and endogenous human proteinases to which the body may be exposed during pathological states (e.g. pancreatitis (trypsin), severe inflammation (HNE and cat G ) ,and sepsis (bacterial proteinases)). Underhomeostatic conditions, most proteolytic activities are tightlyregulated by endogenous inhibitors; however, this is markedly altered at sites of infection and/or inflammation. Two mechanisms are recognized as being responsible for this change. In the first, an oxidative burst of activated phagocytes eliminates most of

the anti-elastase activity due to a-1-PI. This facilitates the second mechanism which involves neutrophil elastase cleavage and inactivation of other serpins (47),including a-1-Achy. The latter event can also easily be executed by pathogenderived proteinases which are generally poorly controlled by endogenous inhibitors, with a-1-Achy being exceptionally sensitive to this type of limited proteolysis. The neutrophil chemoattractant activity of a-1-Achy (actually a-1-Achy’) results only when the native inhibitor is cleaved within the reactive-site loop, proteolysis at other sites having no effect. a-1-PI’ has already been shown to be a human neutrophil chemoattractant (34), and the results shown here for a-lAchy’, together with preliminary data on inactive forms of both AT I11 and C1 inhibitor, indicate that this may be a property of most proteinase-inactivated serpins. Heparin cofactor 11, however, is an exception, possessing neutrophil chemoattractant activity which is located in the amino-terminal region of the native protein and only detected after proteolysis (48). Cleavage of the reactive-site region of this inhibitor does not result in the generation of chemotactic activity. Since all forms of a-1-Achy’ have equal chemotactic activity, regardless of the position of peptide bond cleavage within the reactive-site loop which leads to their formation, it is likely that the exposure ofnew sites on the surface of the modified protein resulting from an S to R shift after peptide bond cleavage is the mechanism utilized in the generation of this activity rather thanregions within the reactive-site loop. Thisis supported by the fact that the carboxyl-terminal peptide arising by enzymatic cleavage is apparently re-folded into a @-pleated sheetstructure and at least partially buried within the modified protein (29, 30). It has been suggested, however, that the carboxyl-terminal peptide of a-1-PI, generated by enzymatic inactivation with macrophage elastase (34), is responsible for chemotactic activity, and this hasbeen partially confirmed in the case of a1-PI where the amino acid sequence directly involved in recognition of the specific receptor for serpin inhibitory complexes or cleaved inhibitors was pinpointed as a pentapeptide domain residing downstream from the reactive site (amino acid residues 370-374 (Phe-Val-Phe-Leu-Met))(49, 50). It is highly possible that a similar neodomain is involved in chemotaxis, not only by a-1-PI’ but also by a-1-Achy’, since in the serpin superfamily this particular region is highly conserved. Such a suggestion is supported by data presented here which show that a-1-Achy’ and a-1-PI’ can block the chemotactic effects of each other, indicating that they probably interact with the same receptor on neutrophil cell surfaces, although it is difficult to understand how this can happen if, in both cases, the carboxyl-terminal peptide is buried within the modified protein. One possible explanation which has been suggested is that the dynamic structure of cleaved or complexed serpins in solution may differ slightly from those obtained through crystallographic analysis (50). The chemotactic activity of a-1-Achy. may have great physiological importance, since it provides a mechanism for the attraction of neutrophils to inflammatory sites. However, it should be pointed out that the same receptors used by C5a could still be involved in a-1-Achy’ binding, although this has not been investigated by us. In any case, the activity which we describe here is almost certainly important in the lung where studies of bronchial secretions of patients with chronic bronchitis have shown that the concentrations of a-1-Achy are higher than predicted by simple diffusion from blood plasma (51, 52). This suggests that a-1-Achy may be synthesized within the lung, possibly by alveolar macrophages (53).

21487

Activity Chemotactic Anti-chymotrypsin However, it has been recently shown that lung secretions containing a-1-Achy are low in inhibitory activity against chymotrypsin-like proteinases (54). This may indicate that a portion of the inhibitor has been proteolytically inactivated and, therefore, is partially responsible for the accumulation of neutrophils within the lung and in bronchial secretions in inflammatory states (55). The rate of elimination of a-1-Achy' from the blood was studied to determine whether it is removed from the circulation by the same receptor which eliminates a - l Achy-proteinase complexes (56). Such a rapid removal would presumably decrease its effectiveness as a neutrophil chemotactic factor. This was not thecase, however, the clearance of the modified inhibitor being essentially identical with that of native a-1-Achy and consistent with that found for cleaved forms of AT 111, a-1-PI, and a-2-antiplasmin (25, 57). With the exception of the latter inhibitor, each of these serpins in complex with its target proteinase is removed from the circulation by the same pathway (31-33, 56) utilizing a hepatic receptor referred to as serpin receptor 1 (56). However, a-2antiplasmin-proteinase complexes are eliminated by a separate pathway utilizing serpin receptor 2 (31-33). Since a-1Achy' is not apparently recognized by serpin receptor 1, this receptor must be different from that utilized for the generation of neutrophil chemotaxis. Indeed, thedata obtained confirm the distinct structural differences which must exist between modified and proteinase-complexed serpins (25) because both forms of a-l-PI are chemotactic (34, 58) but only the complex is rapidly removed through a specific receptor. Finally, it is likely that these results, while being obtained in a murine system, are applicable to humans since both a-1-PI (59) and AT I11 (60) in complex with their target proteinases are rapidly removed from the human circulation. Among members of the serpin family, a-1-Achy would appear to be an excellent candidate for signaling during physiological stress, based on the following reasons. (a) It is extremely sensitive to proteolytic inactivation. (b) Both a-lAchy:cat G complexes and a-1-Achy' can induce IL-6 synthesis and,indirectly, acute phase protein synthesis(47). (c) The plasma elimination time of the modified inhibitor insures a long-lasting stimulatory action on cells producing IL-6. (d) a1-Achy' can act as a neutrophil attractant at inflammatory sites where it would be expected to exist in this form in high concentration and for long periods of time. It is tempting to suggest that permanent stimulation of IL-6 in individuals with malignant disease maybe occurring because of their abnormally high circulating levels of a-1-Achy (61), most of which is in an inactive form: together with other acute phase plasma proteins. Indeed, the tumor cells can themselves act asa source of inactive a-1-Achy (62, 63), the conversion presumably occurring in situ. However, the importance of this mechanism in promoting tumor growth remains to be established. REFERENCES

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