©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Macroglobulin Bait Region Variants
A ROLE FOR THE BAIT REGION IN TETRAMER FORMATION (*)

Peter G. W. Gettins (1)(§), Kwang-ho Hahn (1), Brenda C. Crews (2)

From the (1) Department of Biochemistry, University of Illinois, Chicago, Illinois 60612 and the (2) Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To test the hypothesis that a large portion of the bait region of human -macroglobulin (M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human M variants with truncated bait regions and examined whether these variants folded normally and functioned as proteinase inhibitors. Each variant contains sites that are normal bait region cleavage sites in wild-type M, including the primary trypsin cleavage site. The truncated bait regions are shorter by 23 and 27 residues, respectively, and lack the C-terminal portion as well as different parts of the N-terminal section of the bait region. We found that such bait region truncation permitted normal folding of the monomers as well as formation of the thiol ester and dimerization by disulfide cross-linking, although the resulting species bound 6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more like thiol ester-cleaved M than native M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric M, decoupled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to ``trap'' proteinase. This was despite apparent cleavage of the bait region by proteinase, albeit at very much reduced rates relative to wild-type tetrameric M. The kinetics of thiol ester cleavage-dependent protein conformational changes also changed from sigmoidal to exponential. These findings indicate that residues in the bait region appear to be necessary for noncovalent association of 360-kDa disulfide-linked dimers to give tetrameric M and suggest a role for the bait region in normal M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping.


INTRODUCTION

Human -macroglobulin (M)() is a high molecular mass, tetrameric, broad range plasma proteinase inhibitor (1) that is organized as a noncovalently associated dimer of disulfide-linked dimers (2) . It inhibits proteinases by the unusual mechanism of physically sequestering the proteinase as a result of proteinase-triggered large-scale conformational changes in M and can inhibit up to 2 mol of proteinase/mol of M tetramer, depending on the size of the proteinase (3). The interaction between the proteinase and M that initiates the trapping sequence is a limited proteolytic cleavage of M in a restricted region, termed the bait region. The bait region occurs in all -macroglobulins, but shows great variation in length and amino acid sequence between different -macroglobulin species (1) (Fig. 1). In contrast, the remainder of the polypeptide shows very high sequence similarity between -macroglobulins, including conservation of most of the disulfides (1), suggesting very similar folding of the -macroglobulin monomers.


Figure 1: Bait region sequences of wild-type and variant human M and, for comparison, of PZP, rat M, and rat I using the one-letter amino acid code. The bait region covers approximately residues 666-706 in the human inhibitor. Within this region are all of the primary proteinase cleavage sites (underlined), most of the secondary cleavage sites, and very little sequence homology with M from other species. Outside of this region, the primary structures of M from different species show very high sequence homology. Sites of cleavage in human M by trypsin, chymotrypsin, and porcine pancreatic elastase are indicated by downwardarrows and the letters T, C, and P, respectively. The positions corresponding to the DNA cleavage sites by the NsiI, MluI, and BsiWI restriction endonucleases used in the construction of the variants are indicated. Sequence data for human M, PZP, rat M, and rat I are taken from Sottrup-Jensen et al. (1).



A puzzle regarding the functioning of the trapping mechanism of -macroglobulins is that limited proteolysis anywhere within the bait region results in triggering the same conformational change. Similarly puzzling is that the same mechanism appears to operate in different -macroglobulins despite the large differences in length, sequence, and composition of the bait regions (Fig. 1). H NMR studies on human M have shown that at least parts of the bait region are mobile and surface-accessible (4, 5) . This has led to a model for the organization of M as a structurally conserved scaffold formed by the majority of the polypeptide to which is attached a less structured module composed of the bait region residues (6) . Since the structure of the flexible portion of the bait region does not appear to depend on the conformation of the remainder of the protein (5, 7) , such a model could readily accommodate the large differences between bait regions that occur between different -macroglobulins and suggested to us that it might be possible to shorten a given bait region without loss of inhibitory properties. To test this, we have expressed and characterized two human M variants in which a large portion of the normal bait region sequence from residues 676 to 706 has been removed and much shorter portions substituted, each of which contains sites that are normally attacked by specific proteinases in wild-type M, including the primary trypsin cleavage site (8, 9) . These truncated bait regions are shorter by 23 and 27 residues, respectively.


MATERIALS AND METHODS

Construction of Vectors for Expression of Bait Region Variants of Human -Macroglobulin

The vector for expression of variant 1, pA2M-BRC1, was constructed from the wild-type expression plasmid p1167 (8) . In brief, a 1683-base pair fragment of p1167, covering M residues 146-707, was excised by digestion with the restriction endonucleases BamHI and BsiWI and ligated into M13mp18 containing a modified polylinker region in which the portion of the polylinker between the PstI and SphI sites had been removed and replaced by a synthetic oligonucleotide containing AflII, BsiWI, and XhoI sites (coding strand, 5`-ACTTAAGCGTACGCTCGAGTCATG-3`; noncoding strand, 5`-ACTCGAGCGTACGCTTAAGTTGCA-3`). The phage construct was doubly digested with NsiI and BsiWI to remove the portion of DNA covering most of the bait region. A double-stranded linker (coding strand, 5`-TGGCCTACGCGTGCAC-3`; noncoding strand, 5`-GTACGTGCACGCGTAGGCCATGCA-3`) containing a silent MluI site was then inserted to create a new 1605-base pair BamHI-BsiWI fragment with a modified bait region cDNA sequence. The BamHI-BsiWI fragment was excised and religated into p1167 that had been cut with BamHI and BsiWI to create pA2M-BRC1, which was used for expression of M variant 1. pA2M-BRC2, used for expression of M variant 2, was derived directly from pA2M-BRC1. pA2M-BRC1 was cut with MluI and BsiWI to remove a portion of the bait region cDNA, and a longer double-stranded linker (coding strand, 5`-CGCGTGGGCTTTTACGAGAGC-3`; noncoding strand, 5`-GTACGCTCTCGTAAAAGCCCA-3`) was inserted between the cut ends. The success of the mutagenesis was confirmed by sequencing of the plasmid pA2M species.

Stable Transfection of Baby Hamster Kidney Cells

Stably transfected baby hamster kidney cells expressing wild-type and bait region variants of human -macroglobulin were prepared by cotransfection with plasmids pRMH140 and pSV2dhfr and the appropriate -macroglobulin expression plasmid, with selection for high producers by resistance to methotrexate and neomycin, as described (9) . Typically, 5 µg of pRMH140 and pSV2dhfr and 20 µg of the -macroglobulin plasmid were used. Calcium phosphate precipitation was used for the transfection.

Isolation of -Macroglobulin

Plasma M was isolated from pooled outdated plasma obtained from the Vanderbilt Blood Bank by chromatography on zinc chelate resin, Cibacron blue gel, and AcA22 as described previously (10) . All forms of recombinant M were isolated from serum-free cycles of growth medium using only the zinc chelate resin chromatography step, which yielded material sufficiently pure for further characterization as judged by polyacrylamide gel electrophoresis. Concentrations of all forms of M were determined spectrophotometrically using an extinction coefficient for the plasma protein of 640,000 M cm(11) since the changes in the bait region were expected to have a negligible effect on the absorbance at 280 nm.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gels were run under nondenaturing conditions in 5% acrylamide slabs according to the procedure of Davis (12) or under denaturing conditions on 7.5% gels according to the procedure of Laemmli (13) . Unless otherwise noted, samples for SDS-PAGE were denatured and reduced for 45 min at 37 °C (to prevent heat cleavage (14, 15) ) in buffer containing 1% SDS and 0.8% dithiothreitol. 1 mM phenylmethanesulfonyl fluoride was added to M samples containing proteinase to inactivate the proteinase and thus prevent additional cleavage of M during denaturation. Molecular mass standards were myosin (200 kDa), -galactosidase (116.3 kDa), phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa).

N-terminal Sequencing

Samples for N-terminal sequence analysis were prepared by electroblot transfer of trypsin cleavage fragments from a 5% polyacrylamide gel run under denaturing/reducing conditions to a polyvinylidene difluoride membrane using published procedures (16) . The sequences were determined on an ABI Model 477A protein sequencer in the Protein Sequencing/Synthesis Laboratory at the University of Illinois at Chicago.

Assays

Trypsin trapping assays were carried out as described previously (17) and involved measurement of residual trypsin activity following addition of excess soybean trypsin inhibitor to complex any nontrapped trypsin. Trypsin activity was measured spectrophotometrically with the substrate tosylarginine methyl ester. Assays of protected trypsin were carried out after a 5-min reaction of trypsin for plasma M and after up to a 60-min reaction with trypsin for the variants. Free sulfhydryl groups were determined by spectrophotometric assay on a Shimadzu 2100PC apparatus using 5,5`-dithiobis(2-nitrobenzoic acid) (17) .

Fluorescence Measurements

Wavelength and kinetic measurements of TNS fluorescence were made on an SLM-AMINCO 8000 spectrofluorometer using samples of 1.2 ml in acrylic cuvettes. A TNS concentration of 50 µM was used for all measurements. Excitation was at 316 nm, with slits of 4 nm. Emission slit settings of 4 nm for emission spectra and of 16 nm for monitoring emission at a constant wavelength were used.

Data Analysis

Kinetic data were fitted by nonlinear least squares using the program MINSQ II (MicroMath Scientific Software, Salt Lake City, UT).


RESULTS

Electrophoretic Behavior of Bait Region Variants

To compare the properties of the two variants with those of wild-type M, we examined their electrophoretic behavior on polyacrylamide gels run under nondenaturing conditions. Both variants migrated much faster than wild-type recombinant or plasma M, at a position consistent with their being dimeric species, as judged by comparison with Limulus M, which occurs naturally as a dimer, and with SDS-dissociated wild-type M (Fig. 2, lanesa, c, e, h, and j). When run under denaturing conditions in the presence of dithiothreitol, the differences between the normal and variant forms of M disappeared (Fig. 3), reflecting similar size of the monomers. Plasma and wild-type recombinant M gave a single band corresponding to the M monomer (Fig. 3, lanesb and c). The two M variants also gave single bands at the same position as wild-type recombinant M (Fig. 3, lanesd and e). In the absence of dithiothreitol, all species gave a single band at higher molecular mass, corresponding to the disulfide-linked 360-kDa dimer (data not shown).


Figure 2: Electrophoretic mobilities of wild-type and variant human M and of Limulus M on a 5% polyacrylamide gel run under nondenaturing conditions in the absence and presence of trypsin. pA2M, rA2M, LA2M, BR1, and BR2 refer to plasma M, recombinant wild-type M, Limulus M, variant 1, and variant 2, respectively. The designations ``-'' and ``+'' signify the absence and presence of trypsin. Laneg contains wild-type recombinant M in the presence of 1% SDS to promote dissociation into dimers. Trypsin treatment involved incubation at room temperature for 10 min with 1.1 eq of trypsin/M dimer at an M concentration of 1 mg/ml. The positions of dimer and tetramer are indicated by arrows. Phenylmethanesulfonyl fluoride to a concentration of 2 mM was added to terminate the proteinase reactions.




Figure 3: SDS-PAGE (7.5% gel) of wild-type and variant M run under reducing conditions. Samples were prepared as described under ``Materials and Methods.'' Lanea, molecular mass standards; laneb, plasma M; lanec, wild-type recombinant M; laned, variant 1; lanee, variant 2.



Presence and Properties of Internal Thiol Esters

Although the thiol ester is an intrinsically reactive group, it is kinetically stabilized in the native M structure by being located in a hydrophobic cleft with limited solvent accessibility (6, 18, 19, 20) . The presence of an intact thiol ester in an M variant is thus a sensitive indicator of the correct structure of the variant in the vicinity of the thiol ester, needed both to generate and to stabilize the thiol ester. Both bait region variants were found to contain intact thiol esters as judged by the ability to generate free sulfhydryl groups from them upon incubation with methylamine ().

To compare the properties of the thiol esters in the two variants with those of the thiol esters in normal plasma M, we examined the kinetics of their reaction with methylamine. The rate of reaction of the thiol esters in both M variants, determined by the rate of appearance of free SH groups, was indistinguishable from that in plasma M (Fig. 4). Since the reactivity of the thiol ester in plasma M is determined not only by the nucleophilicity of the attacking base, but also by the accessibility to the attacking nucleophile, this identity between the variants and plasma M suggests very similar or identical local environments.


Figure 4: Time course of free thiol release from plasma and variant M by reaction with 0.2 M methylamine at pH 8. Free thiol concentration was determined by continuous spectrophotometric monitoring of the change in absorbance caused by reaction of the liberated thiol with 5,5`-dithiobis(2-nitrobenzoic acid). , variant 1; , variant 2; , wild-type M. The solidlines represent the least-squares best fits to the data.



Effects of Variants on TNS Fluorescence

TNS fluorescence has been used extensively to follow the protein conformational changes that result from bait region and/or thiol ester cleavage in M (21, 22, 23, 24) . We initially examined the ability of the M variants to bind to and alter TNS fluorescence and compared the effects with those of the well studied human plasma M. In contrast to the relatively modest enhancement produced by plasma M (Fig. 5A), the two variants produced 5-8-fold greater enhancement of TNS fluorescence (Fig. 5, B and C) and a larger blue shift, with at 430 nm, indicative of a TNS-binding site that more closely resembles that of conformationally altered plasma M than that of native M.


Figure 5: Comparison of the changes in TNS fluorescence produced by plasma and variant M before and after reaction with methylamine. A, plasma M; B, variant 1; C, variant 2. In each panel, spectruma is of unreacted M, and spectrumb is of methylamine-reacted M. The TNS concentration was 50 µM in each case, and the concentration of dimeric M was 0.4 µM.



Conformational Changes following Reaction of Variants with Methylamine

The mobility of tetrameric human plasma M or dimeric Limulus M on polyacrylamide gels run under nondenaturing conditions can be used to distinguish between native and methylamine-treated or proteinase-complexed M (14, 25) . The increase in mobility between native and methylamine-treated or proteinase-reacted forms has led to the designation of native M as the ``slow'' form and reacted M as the ``fast'' form (14) and has been correlated with a compaction of the molecule (14, 26) . The two dimeric variants showed imperceptible changes in mobility following reaction with methylamine, compared both with tetrameric human M and dimeric Limulus M (Fig. 6), suggesting that any conformational change resulting from thiol ester cleavage is different both in magnitude and kind from that which occurs in the normal tetramer or even in a fully functional dimer.


Figure 6: Effect of thiol ester cleavage on electrophoretic mobilities of plasma and variant human M and Limulus M run under nondenaturing conditions on a 5% polyacrylamide gel. The designations ``-'' and ``+'' signify that M was untreated or reacted with methylamine, respectively. ``S'' and ``F'' indicate positions of slow and fast conformers, respectively. For plasma M and for the two variants, methylamine-treated samples were prepared by reaction with 0.2 M methylamine at pH 8.0 for 1 h, which is sufficient to ensure complete cleavage of the thiol esters. For Limulus M, which reacts with methylamine more slowly, reaction was with 0.4 M methylamine at pH 8.0 overnight.



As a second way of comparing the thiol ester cleavage-induced conformational change in the two variants with that in plasma M, we examined the effect that these species had on the fluorescence emission spectrum of TNS. Both variants gave a small increase in fluorescence intensity, although without an alteration in the position of the maximum, whereas plasma M gave a 4-fold increase in intensity and a shift in the emission maximum of 40 nm (Fig. 5).

We also examined the kinetics of the small methylamine-induced protein conformational change by monitoring TNS fluorescence changes and found a major difference between the variants and plasma M. In plasma M, cleavage of two thiol esters, presumably within the dimer that constitutes a proteinase-binding site, is required before any large-scale conformational change occurs in that dimer (21) . This cooperativity leads to sigmoidal kinetics of methylamine-induced conformational change that can be well fitted to a model of initial reaction of the thiol esters within a half-molecule (S and S) (with rate constant k`) and subsequent slow conformational change (with rate constant k ) once both thiol esters within a given half of the tetramer have been cleaved (S and S) (Fig. S1). (Note, however, that more complex schemes could be envisaged involving additional steps. Indeed, a more complex scheme is necessary to explain sigmoidal kinetics for the monomeric thiol ester-containing protein C3 upon thiol ester cleavage (27) . Also note that the two subunits in tetrameric plasma M that constitute a proteinase-binding site and that are presumably the ones that change conformation cooperatively upon thiol ester cleavage of both subunits may well be from different disulfide-linked half-molecules rather than the same one.)


Figure S1: Scheme 1.



Using the change in TNS fluorescence that accompanies this change in protein conformation, we were able to reproduce the sigmoidal behavior for plasma M (Fig. 7C) and to obtain a rate constant for the protein conformational change (9 10 s) very similar to the published value (10 10 s) (21) . The behavior of the variants was different and simpler. No sigmoidal kinetics were observed. Instead, the TNS-monitored conformational change was well described by a single exponential, giving a pseudo first-order rate constant (1.9 10 s) similar to that of thiol ester cleavage (Fig. 7, A and B). Thus, the protein conformational change in the two variants, which is a different change from wild-type M, did not depend on cleavage of both thiol esters within the same disulfide-linked half-molecule.


Figure 7: Time course of protein conformational changes resulting from reaction of 0.4 µM M species with 0.2 M methylamine at pH 8.0 followed by changes in TNS fluorescence. A, variant 1; B, variant 2; C, plasma M. The solidlines represent the least-squares fits to a pseudo first-order reaction for variants 1 and 2 and to a two-step conformational change described in Scheme 1 for plasma M (21). Using the sigmoidal fit for the variants gave a very much poorer fit than using the single exponential.



The ability of each of the two variants to protect trypsin from inhibition by soybean trypsin inhibitor through sequestration of the proteinase was determined by reaction of the variant with trypsin, addition of excess soybean trypsin inhibitor, and assaying for residual trypsin activity. Although plasma M was capable of protecting proteinase in this assay, neither of the variants showed any significant trypsin protection ability after incubation for either 5 or 60 min with trypsin.

Proteolytic Reaction of Bait Region Variants

To determine whether the M variants retained an exposed flexible bait region that remained the preferred site of proteolytic cleavage, we examined the reaction of each of the variants with proteinase. Reaction with trypsin resulted in cleavage of both variants as judged by the disappearance of the 180-kDa monomer band and the appearance of cleavage products on SDS-PAGE (Fig. 8). Although there was evidence for cleavage in the bait region, cleavages at additional sites made interpretation of the degradation patterns problematical. The rate of cleavage of the bait region of each variant by trypsin was very much less than for plasma M, and the reaction was catalytic rather than stoichiometric. For variant 2, bait region cleavage bands were the first formed, but prolonged incubation resulted in the appearance of smaller fragments with the consequent disappearance of the higher molecular mass bait region cleavage bands (data not shown). For variant 1, bait region cleavage occurred, but another major cleavage site resulted in only a small reduction in size of the 180-kDa band and the appearance of a slightly smaller intense band. These findings can be understood in terms of cleavage within the bait region still being possible in both variants, but at such a reduced rate that secondary cleavages outside of the bait region also become important. As a result, further degradation of the variants can occur, particularly with longer incubations. The reduced rate of reaction of proteinase with the shortened bait regions must reflect reduced accessibility or flexibility of the bait region peptide as a result of removal of flanking residues on the N- and/or C-terminal sides.


Figure 8: Effect of incubation of plasma and variant M with trypsin for 5 min as analyzed by SDS-PAGE (7.5%). pA2M, BR1, and BR2 refer to plasma M, variant 1, and variant 2, respectively. The designations ``-'' and ``+'' signify the absence or presence of trypsin. Trypsin treatment involved incubation of 2.8 µM M dimer with 3.0 µM trypsin at room temperature for 5 min. The positions of the bait region cleavage bands (95-85 kDa) and intact monomer bands (180 kDa) are indicated by arrows. Phenylmethanesulfonyl fluoride to a concentration of 2 mM was added to terminate the proteinase reactions.



To confirm that the bait region remained a major site of proteolysis, N-terminal sequencing was carried out on the dominant cleavage bands of variant 2 after reaction with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (see Fig. 8for appearance of the gel). Since individual bands were not resolved in this region, the diffuse stained region around 90 kDa was cut into two parts (upper and lower) and separately sequenced. The normal N-terminal sequence SVSGK was found to be a major component of the lower and a minor component of the upper part of the band. A new N terminus, with the sequence VGFYE and corresponding to cleavage at the expected bait region trypsin cleavage site, was found to be a major component of the upper and a minor component of the lower part of the band. In both the upper and lower parts, another N-terminal sequence, AFQPF, was also found. This corresponds to secondary cleavage near the bait region at residue 765 (normal M numbering) and may reflect greater accessibility of this region in the absence of dimer-dimer interactions.

Proteinase-induced Conformational Change

We examined the nature of the proteinase-induced conformational changes in the two variants both by PAGE under nondenaturing conditions and by TNS fluorescence changes. Trypsin reaction with both variants resulted in a different change in electrophoretic mobility from that with both wild-type tetrameric human M and dimeric Limulus M. Whereas plasma and wild-type recombinant human M as well as Limulus M showed the characteristic increase in mobility upon reaction with proteinase (Fig. 2, lanesb, d, and f), both variants showed a slight decrease in mobility (lanesi and k).

The TNS fluorescence spectra also showed different changes for the M variants upon reaction with proteinase compared with plasma M. Thus, both variants produced a decrease in TNS fluorescence intensity with little change in upon reaction with proteinase, whereas plasma M gave a large increase in intensity and a blue shift of 40 nm in (data not shown).

Coupling between Bait Region Cleavage and Thiol Ester Activation

In plasma M, bait region cleavage by proteinase results in limited conformational change that alters the environment of the thiol ester and makes it much more reactive to nucleophilic attack, generating the so-called ``nascent state'' (28) . Cleavage of both thiol esters within a half-molecule is then the trigger for the large-scale conformational change that results in proteinase trapping (21) . There is thus a necessary coupling of bait region cleavage and thiol ester activation for the proteinase trapping mechanism to occur in plasma M. To determine whether such coupling still occurred in the dimeric M variants, we assayed proteinase-treated M for free SH release. Even after 1 h of incubation with 1.1 eq of trypsin/half-molecule, no free SH groups were detectable by continuous 5,5`-dithiobis(2-nitrobenzoic acid) assay, in contrast to the behavior of plasma M (). That the thiol esters were still present and accessible to nucleophile was demonstrated by the subsequent ability of methylamine to release free SH groups (), at a rate 6 times faster than in non-proteinase-treated variants.


DISCUSSION

In this study, we tested the hypothesis that portions of the bait region of M can be removed without adversely affecting the properties of M by examining the properties of two bait region-truncated human M variants to see whether these truncations affected the correct folding of the protein and the ability of M to function as a proteinase inhibitor. The findings proved to be both more complex and more interesting than anticipated due to unexpected effects of the bait region truncation on tetramer formation. Thus, removal of 23 or 27 of the 40 bait region residues apparently still resulted in correct folding of the M monomer and normal dimerization via intermolecular disulfide formation, but prevented noncovalent association of the disulfide-linked half-molecules to give tetrameric M. The dimers were incapable of protecting trypsin from soybean trypsin inhibitor by trapping or of significantly activating the thiol esters toward nucleophilic attack and reacted much more slowly than the wild type with proteinase. This inability to trap proteinase may be due to one of several causes: (i) the uncoupling of bait region cleavage from thiol ester activation; (ii) the failure to form tetramer; or (iii) the interrelationship of both of these processes, namely the requirement of parts of the bait region for tetramer formation, which in turn is needed for the coupling of bait region cleavage to thiol ester activation and of thiol ester cleavage to proteinase-trapping conformational change. These unexpected findings should provide new insights into the mechanism of activation of the thiol ester by bait region proteolysis.

Correct Folding of Monomers

Correct folding of the monomers was indicated by several properties of the variants. Thus, (i) the thiol ester bond formed in each variant was stable in the absence of nucleophile and reacted with methylamine at a rate comparable to that of wild-type M to give close to the expected number of free SH groups. Our understanding of thiol ester formation in M to give functional slow form M is that it occurs after the correct folding of the M polypeptide to give a form resembling fast form M in which the cysteine and glutamine residues (positions 949 and 952, respectively) are correctly positioned and in a suitable environment for formation of a stable thiol ester by elimination of NH (9, 29). Thus, both M variants must provide favorable folded structures for formation of the thiol ester linkage similar to those in wild-type M. However, it should be noted that the effect of these variants on TNS fluorescence was more like that of conformationally altered M rather than native M, so that the overall conformation of correctly folded domains may not be identical to that in wild-type M. (ii) Wild-type M is composed of pairs of disulfide-linked dimers (2) . Formation of disulfide-linked dimer rather than higher molecular mass multimers must require correct folding of the appropriate domains such that these two cross-links can form. Both on native gel electrophoresis and SDS-PAGE under nonreducing conditions, the two variants migrated as covalently linked dimers, which dissociated into 180-kDa monomers upon reduction. (iii) Other evidence for correct folding is the immunological cross-reactivity of the variants with the polyclonal antibody used in the radial immunodiffusion assay and the normal behavior of the variants during the zinc chelate affinity chromatography step that is used for isolation of tetrameric human plasma M.

Involvement of the Bait Region in Tetramer Formation

Although we had expected correct folding of the monomers and intersubunit disulfide bonding to give dimers, we had not anticipated that these dimers would fail to form tetramers by noncovalent association. A common feature of the variants was that a large part of the C-terminal region was missing in each case, including the portion that does not contain principal proteolysis sites in the wild-type inhibitor (residues 701-705). It thus appears that some or all of the C-terminal portion may be involved in the noncovalent association of M dimers to give tetramers. Perhaps relevant to the role of the bait region in interdimer interactions is that it is much harder to disrupt the noncovalent interactions in proteinase-reacted M than in native M, suggesting a major alteration in the interdimer contacts (30, 31) . Although this could be another consequence of the global conformational changes in M that occur upon bait region and thiol ester cleavage, it should be noted that fluorescence studies have localized both the thiol ester and the bait region to the central region of M (32, 33) , which is the region where the noncovalent interdimer contacts appear to occur (34) . This localization of the thiol ester has more recently been confirmed by electron microscopy studies (35, 36) .

If noncovalent contacts between the C-terminal portion of the bait region of a monomer of one dimer and regions on a second dimer are critical for tetramerization, it might be expected that major differences in this C-terminal region might parallel different oligomerization states in other -macroglobulins. This appears to be the case. Thus, human PZP, which has a much longer bait region than human M (1, 37) , is a predominantly disulfide-linked dimer in the native state and has the sequence SSGPVP immediately preceding the conserved ETXR sequence, whereas a very different sequence, VEEPHT, is found in human M (Fig. 1). Upon reaction with proteinase, but not methylamine, the equilibrium between dimer and tetramer shifts markedly toward tetramer (38) , indicating an alteration in the nature of the noncovalent dimer-dimer interactions following bait region cleavage and thus possibly involving the bait region directly. The bait regions of the two rat I variants are even more different from human M than is PZP, having a large number of prolines in the C-terminal region of the bait region (8 out of 18 residues) (Fig. 1). These I proteins also differ in oligomerization state from human M in being monomeric.

What Constitutes a Proteinase Trap?

Tetrameric human M can inhibit up to 2 mol of proteinase. However, it has still not been definitively shown whether two subunits from the same or different covalent dimers form a proteinase-binding site (Fig. 9). Analysis of the pattern of proteinase-M covalent cross-links favors a side-to-side mode of noncovalent association of covalent dimers (Fig. 9B), and electron microscopy studies of the difference in density between methylamine- and proteinase-treated M indicate that the upper and lower halves of the M ``H'' represent proteinase-binding sites (39) . Thus, the schematic representation in Fig. 9B most probably represents the mode of proteinase inhibition in human M. The simplest explanation for the failure of either of the variants to trap proteinase is therefore that a trap could only exist in the M tetramer as a result of the bringing together of the separate halves of the trap from different covalent dimers.


Figure 9: Schematic representation of two ways in which the two disulfide-linked dimers of plasma M might interact noncovalently to give tetrameric M and to form the two proteinase-binding sites of the tetramer. A, formation of a proteinase-binding site by a disulfide-linked dimer; B, formation of a proteinase-binding site by two monomers, one from each of two noncovalently associated disulfide-linked dimers. In A and B, one of the disulfide-linked dimers is shown hatched. No attempt has been made to indicate the position of the intersubunit disulfide bonds or the position of the bait regions. However, we propose that the bait regions lie in the contact region between a hatched and a nonhatched dimer. C and D represent the type of half-molecules that would result from failure of the dimers to associate noncovalently for cases A and B, respectively. Note how proteinase sequestration (i.e. trapping) is only possible in C. The circles labeled P represent trapped proteinase that is not free to dissociate from complex with M.



Although functional dimers exist in other species, e.g.Limulus, there is no requirement that these dimers be equivalent to the disulfide-linked dimer within the human M tetramer. Rather, the Limulus dimer may resemble the two noncovalently associated monomeric units of the M tetramer that we propose form a trap, with the difference that in Limulus these are the subunits that are covalently linked. In keeping with a structural difference between Limulus covalent dimers and human M covalent dimers (represented by the present bait region variants) is that the Limulus dimer undergoes an increase in electrophoretic mobility upon reaction with proteinase (analogous to tetrameric human M), whereas the M variant dimers showed a small decrease in electrophoretic mobility (Fig. 2). In addition, the dimeric M variants behave quite differently toward TNS binding than would be expected from them simply acting as one-half of an M tetramer.

Another consideration in the failure of the variants to trap proteinase is that the rate of proteinase reaction is very much slower than for wild-type M. However, it is not the rate of reaction that is important, but the rate of conformational change following bait region cleavage that determines the efficiency of proteinase sequestration. Thus, rapid reaction followed by a very slow trapping conformational change could result in less trapping than a slow proteolysis followed by a rapid conformational change.

Uncoupling of Bait Region Cleavage and Thiol Ester Activation and Hydrolysis

An alternative explanation for the failure of the variants to trap proteinase is that bait region cleavage did not result in significant activation of the thiol ester toward nucleophilic attack. Thus, no free SH groups were generated after reaction of the variants with trypsin, although there was a small increase in the rate of methylamine cleavage of the thiol esters in trypsin-treated M variants (6-fold faster than in the native proteins). This, however, is a very much smaller enhancement in reactivity than is normally found in plasma or wild-type recombinant M, where cleavage of the thiol ester is 90% complete within 15 s after reaction with proteinase (40) . The absence of the appropriate conformational change after proteolytic cleavage is, however, more likely to be due to the absence of cooperative interactions between noncovalently associated subunits than to the uncoupling of thiol ester activation and bait region cleavage. Thus, direct cleavage of the thiol esters with methylamine, although proceeding at a normal rate, failed to bring about the normal conformational change and did not show the cooperativity that is found in tetrameric M (Fig. 7). It is therefore more likely that the uncoupling of thiol ester activation from bait region cleavage and the absence of cooperative thiol ester cleavage-dependent protein conformational change in the variants result from the same cause, namely the absence of noncovalent dimer-dimer interactions.

Uncoupling of bait region cleavage and thiol ester activation has been reported in human M that had been cross-linked between dimeric subunits by cis-platinum. Reaction of this cross-linked species with trypsin gave bait region cleavage without thiol ester activation or conformational change. Consequently, no trapping of proteinase occurred until the cross-links were removed (41). Reaction with methylamine did, however, result in a conformational change since the dimer-dimer interface was present (42) . There may thus be a parallel between such cross-linked M species and the dimeric M variants of the present study in that particular dimer-dimer interactions may be needed both for thiol ester activation and for the conformational change that follows thiol ester cleavage. In the cross-linked tetramer, the bait region-thiol ester linkage may be disrupted by covalently locking the dimer-dimer interface, whereas in the dimeric variants, the interactions are not achievable because tetramers do not form. Thus, the need for tetramer formation may be 2-fold: first, to physically constitute a potential trap by bringing together the two halves of the trap located in different disulfide-linked half-molecules; and second, to provide the changes in dimer-dimer interactions consequent to bait region cleavage that are necessary for thiol ester activation and for linking thiol ester cleavage in both halves of the trap to conformational change. Either of these on its own is, however, sufficient to account for the failure of the present variants to trap proteinase.

Choice of Bait Region Variants

Our choice of bait region variants was originally guided by the expectation that the whole of the bait region outside of the conserved regions is a discrete domain that is not involved in interdimer interactions and that major deletions could be made that would only affect proteinase specificity. The variants that we constructed were thus appropriate for testing bait region requirements for accessibility to, and specificity for, proteinase, but not appropriate for delineating the function of the bait region in maintaining the tetrameric, and therefore functional, state of the inhibitor. Consequently, we plan to make further bait region variants in which (i) selected portions of the C-terminal region of the bait region will be either added back to the present variants or deleted from the wild type and (ii) the remainder of the deleted N-terminal portion of the bait region will be restored. This will allow us to determine whether there are variants that remain dimers but are more rapidly cleaved in the bait region compared with the present variants and also to determine which residues are needed for tetramer formation.

  
Table: Thiol ester cleavage of recombinant M as a result of treatment with methylamine, trypsin, or trypsin followed by methylamine

The values reported are based on the measured change in absorbance. All measurements are the average of three separate determinations. The accuracy of the measurements is estimated to be ±0.1 SH/monomer.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD28187. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 1819-1853 West Polk St., Chicago, IL 60612. Tel.: 312-996-5534; Fax: 312-413-8769.

The abbreviations used are: M, -macroglobulin(s); PAGE, polyacrylamide gel electrophoresis; TNS, 6-(p-toluidino)-2-naphthalenesulfonic acid; PZP, pregnancy zone protein.


ACKNOWLEDGEMENTS

We thank Dr. Assaf Steinschneider for carrying out the N-terminal peptide sequencing, Dr. Esper Boel for the generous gift of the wild-type M expression plasmid p1167, and Dr. Steven T. Olson for critical comments on the manuscript.


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