©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sodium Dodecyl Sulfate-stable Complexes between Serpins and Active or Inactive Proteinases Contain the Region COOH-terminal to the Reactive Site Loop (*)

S Christensen (1), Zuzana Valnickova (1), Ida B. Th (1), Salvatore V. Pizzo (1), Henrik R. Nielsen (2), Peter Roepstorff (2), Jan J. Enghild (1)(§)

From the (1)Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 and (2)Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

ABSTRACT
INTRODUCTION
Experimental Procedures
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recently inhibitors of the serpin family were shown to form complexes with dichloroisocoumarine (DCI)-inactivated proteinases under native conditions (Enghild, J. J., Valnickova, Z., ThI., and Pizzo, S. V.(1994) J. Biol. Chem. 269, 20159-20166). This study demonstrates that serpin-DCI/proteinase complexes resist dissociation when analyzed in reduced SDS-polyacrylamide gel electrophoresis. Previously, SDS-stable serpin-proteinase complexes have been observed only between serpins and catalytically active proteinases. The stability of these complexes is believed to result from an acyl-ester bond between the active site Ser of the proteinase and the -carbonyl group of the scissile bond in the reactive site loop. We have further analyzed the structure of the SDS-stable serpin-proteinase and serpin-DCI/proteinase complexes. The results of these studies demonstrate the presence of the COOH-terminal region of the serpin in both complexes. Since (i) modification of Ser does not prevent formation of SDS-stable complexes and (ii) COOH-terminal peptides are present in both complexes, the previously described mechanism does not sufficiently explain the formation of SDS-stable complexes.


INTRODUCTION

Serpins are the predominating serine proteinase inhibitors in mammals and regulate the serine proteinases involved in blood coagulation, fibrinolysis, complement activation, and inflammation(1, 2) . Although the biological role of many serpins is well understood, the serpin mechanism of proteinase inhibition remains unclear. In contrast, small serine proteinase inhibitors from the kunin and kazal families operate by a well characterized ``standard mechanism''(3) . Several hypotheses describing the interactions between serpins and proteinases have been presented(4, 5, 6, 7, 8, 9, 10, 11, 12) . These hypotheses are based on the assumption that serpins contain an exposed loop, similar to the RSL()of the standard mechanism inhibitors, but vary in the way they predict how the RSL is held in an inhibitory conformation to achieve tight binding inhibition.

Analysis of most serpin-proteinase complexes by SDS-PAGE reveals the presence of an SDS-stable complex formed between the serpin and the proteinase. These complexes are generally believed to be a result of the SDS-induced distortion of the catalytic triad (Ser, His, Asp),()which prevents the deacylation step necessary for dissociation. An ester bond, not present in the native complex, is thought to be formed between O of Ser and the -carbonyl of the scissile bond(13, 14, 15) . Consequently, the RSL is cleaved, releasing the COOH-terminal peptide of the serpin. However, the structures of these SDS-stable complexes have never been characterized and no direct evidence for the presence of this bond has been presented. Since one of the deviations between serpins and standard mechanism inhibitors is the ability to form SDS-stable complexes, understanding the mechanism responsible for their formation might provide valuable hints about the nature of the serpin mechanism of inhibition.

In this study we show that active site modified proteinases are capable of forming SDS-stable complexes with serpins. Moreover, SDS-stable complexes between serpins and active or Ser-modified proteinases are shown to contain the COOH-terminal region of the serpin, indicating that the P-P peptide bond is intact in both complexes. Thus, other cross-links than the acyl-ester bond between the O of Ser and the -carbon of the scissile bond account for the stability of serpin-proteinase complexes.


Experimental Procedures

Materials

DCI and porcine trypsin were from Sigma. Recombinant PI was from Cooper Laboratories, Mountain View, CA. HNE was a kind gift of Drs. Weislaw Watorek and Jan Potempa, University of Georgia, Athens, GA. ACT and cathepsin G (cat G) were from ART Biochemicals, Athens, GA.

SDS-PAGE

Proteins were analyzed in 5-15% linear gradient gels using the buffer system of Bury(16) .

Automated Edman Degradation

Automated Edman degradation was carried out in an Applied Biosystems model 477A sequencer with on-line analysis of the phenylthiohydantoins using an Applied Biosystems model 120A HPLC apparatus. Some samples were transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting before Edman degradation (17).

Preparation of PI- and ACT-Proteinase Complexes

PI or ACT (3 µg) were incubated in 100 mM HEPES, pH 8.0, with increasing amounts of proteinase for 5 min at 25 °C, followed by addition of DCI to a final concentrations of 0.25 mM. Complexes between PI or ACT and DCI-inactivated proteinases were prepared by preincubating HNE, trypsin, or cat G with 0.25 mM DCI in 100 mM HEPES, pH 8.0, for 15 min at 25 °C. Following inactivation PI or ACT (3 µg) were incubated with increasing amount of the appropriate DCI-inactivated proteinase for 30 min at 37 °C. The concentrations of both active and DCI-inactivated proteinases were varied between 0- and 2-fold (HNE), 0- and 3-fold (trypsin), or 0- and 3.5-fold (cat G) molar excess. All samples were reduced with 10 mM dithiothreitol (DTT) and boiled 2 min in SDS-PAGE sample buffer prior to electrophoresis.

Matrix-assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry of Purified Serpin-Proteinase Complexes

Native and inactive complexes were formed as described above followed by reduction with DTT and alkylation with a 2-fold molar excess of iodoacetic acid in 8 M guanidinium HCl. The complexes were separated from unbound proteinase and autolytic fragments by reverse phase HPLC on a RP C2/C18 column in a SMART system (Pharmacia Biotech Inc.) and analyzed by SDS-PAGE, Edman degradation, and mass spectrometry (MS). Mass spectra were acquired in a prototype linear time-of-flight mass spectrometer (Applied Biosystems, Uppsala, Sweden) equipped with a 0.7-m flight tube and a 337 nm nitrogen laser. Spectra were sampled at 300 MHz using -cyano-4-hydroxycinnamic acid as matrix.

Edman Degradation of COOH-terminal CNBr Peptides Derived from PI-DCI/Trypsin

Complexes between PI and trypsin and DCI/trypsin were subjected to SDS-PAGE and transferred to a PVDF membrane. The 65-kDa PI-trypsin or PI-DCI/trypsin complexes were excised and incubated in HCl:methylsulfide for 5 min at 23 °C to convert potential methionine sulfoxide residues to methionine. The PVDF membrane-bound sample was treated with CNBr overnight to cleave Met-Xaa peptide bonds. The resulting CNBr fragments were subsequently subjected to two or three cycles of Edman degradation. In cycle 3 or 4, orthophthaldialdehyde (OPA) was applied to block all non-Pro NH termini. Edman degradation was subsequently continued on fragments with available NH-terminal Pro residues.


RESULTS

SDS-stable Complexes between PI and Active or DCI-inactivated HNE

Inactivation of HNE, trypsin, and cat G was performed as described in Ref. 18. Titrations of PI with active HNE (Fig. 1A) and DCI/HNE (Fig. 1B) were analyzed by reduced SDS-PAGE. All the reaction products were identified by Edman degradation following electrotransfer to PVDF membranes. The formation of the usual 65-kDa PI/HNE complex was almost complete at a 1:1 molar ratio, and only small amounts of modified PI were observed in the presence of excess HNE (Fig. 1A, lanes 4-9). Two distinct P-P-modified forms of PI (40 and 41 kDa) can be distinguished in lanes5 and 6. In the lower band additional cleavage was identified at the Thr-Asp peptide bond in PI. At higher HNE concentrations (Fig. 1A, lanes 7-9), a complex of 54 kDa is observed. This smaller PI/HNE complex was generated as a result of a cleavage of the Ala-Gln peptide bond in the HNE moiety releasing a 11.6-kDa NH-terminal fragment.


Figure 1: SDS-PAGE of complexes between serpins and active or inactive proteinases. PI (3 µg) was titrated with HNE (panelA) or DCI/HNE (panelB) and analyzed by SDS-PAGE. Lane1, PI; lanes 2-9, molar ratios of HNE:PI or DCI/HNE:PI (0.2, 0.6, 0.8, 1.0, 1.2, 1.6, 1.8, and 2.0, respectively); lane10, size marker. In panels C and D, titrations are shown of PI with trypsin and DCI/trypsin, respectively. Lane1, PI; lanes2-9, molar ratios of trypsin:PI or DCI/trypsin:PI (0.25, 0.50, 1.0, 1.2, 1.5, 2.0, 2.5, and 3.0, respectively); lane 10, 4 µg of DCI/trypsin. In panelE and F, titrations are shown of ACT with cat G and DCI/cat G, respectively. Lane1, ACT; lanes 2-9, molar ratios of cat G:ACT or DCI/cat G:ACT (0.29, 0.58, 1.17, 1.47, 1.76, 2.35, 2.93, and 3.5, respectively); lane10, 4.75 µg of active cat G.



A concentration-dependent formation of SDS-stable complexes between PI and DCI/HNE was observed (Fig. 1B). In contrast to the reaction between PI and active HNE, some virgin PI was observed even in the presence of high concentrations of DCI/HNE (Fig. 1B, lanes 7-9). However, we detected no COOH-terminal PI peptide and only insignificant amounts of the 54-kDa complex. The latter complex is due to a autoproteolytic cleavage of HNE before the DCI inactivation. These results demonstrate that, like active HNE, DCI/HNE forms an SDS-stable complex with PI.

SDS-stable Complexes between PI and Active or DCI-inactivated Trypsin

To study if the ability of DCI/HNE to form SDS-stable complexes with PI was restricted to HNE, we analyzed the interaction between PI and trypsin. A titration of PI with active trypsin demonstrated the 65-kDa PI-trypsin complex (Fig. 1C). As seen with HNE, a 54-kDa intermediate complex is formed with excess proteinase. Several closely spaced bands can be discerned due to various cleavage events both in the trypsin and PI moiety. Edman degradation of the smallest intermediate band revealed two NH-terminal sequences starting at Thr in the PI and at the Ser in the trypsin moiety. The 14-kDa double band was apparent in all lanes, including the trypsin control (Fig. 1C, lane 10), and represents autoproteolytic fragments of trypsin. Below the 14-kDa double band, small amounts of the 4.1-kDa COOH-terminal PI-peptide are apparent (Fig. 1C, lanes8 and 9).

Similar to DCI/HNE, a complex is formed between PI and DCI/trypsin (Fig. 1D). Complexes of 54 kDa are observed as well and are probably due to fragmentation of the trypsin molecule before DCI inactivation, as described above for the HNE. As expected, we did not detect modified PI or COOH-terminal peptide in SDS-PAGE. These results show that formation of SDS-stable complexes between PI and DCI-inactivated proteinases is not a property of DCI/HNE alone.

SDS-stable Complexes of ACT and Active or DCI-inactivated Cat G

To further investigate the ability of serpins to form SDS-stable complexes with active and inactive proteinases, we examined the interaction between ACT and cat G. The reaction products are similar to those observed between PI and trypsin (Fig. 1E). The Asn-Gln bond of ACT was cleaved in the 95-kDa complex between ACT-cat G. Two intermediate complexes are readily formed when an excess of cat G is used (Fig. 1E, lanes 4-9). The upper dominant 86-kDa complex between ACT and active cat G was cleaved between residues Leu-Gln in the cat G moiety and between the Asn-Gln peptide bond in the ACT moiety. Several polypeptides below 30 kDa were observed. By comparing active cat G run alone (Fig. 1F, lane10) and DCI/cat G (Fig. 1E, lane10), these were judged to be degradation products of cat G. In Fig. 1F a titration of ACT with DCI/cat G is shown. Again, a concentration-dependent formation of SDS-stable complexes between a serpin and an inactive proteinase was demonstrated. However, complex formation was less pronounced using this pair of serpin-DCI/proteinase but still significant.

Comparison of HNE-PI and DCI/HNE-PI Complexes

The complex between PI and DCI/HNE is slightly larger (Fig. 2, lane3) than the complex between PI and active HNE (Fig. 2, lane2). If the postulated cleavage of the P-P peptide bond in the PI/HNE complex occurs, the 4.1-kDa COOH-terminal peptide is released from the complex following SDS-PAGE. However, the observed mass difference between the two complexes is minute and not explained by the absence of a 4.1-kDa polypeptide from the PI/HNE complex. Edman degradation of both complexes, following transfer to PVDF membranes, demonstrated that the Thr-Asp peptide bond in the PI/HNE complex had been cleaved. The NH termini of the PI-DCI/HNE complex were intact. Proteolysis of Thr-Asp releases a 1.1-kDa peptide and explains the size difference between the two complexes (Fig. 2). This indicates that SDS-stable PI/HNE complexes contain the COOH-terminal peptide. Consequently, the stability of this complex cannot be due to an acyl-ester bond between the active site Ser and the -carboxyl of the P residue, since this would require a nucleophilic attack at the P-P bond followed by deacylation and release of the COOH-terminal peptide.


Figure 2: Comparison of the PI/HNE and PI-DCI/HNE complexes. Complexes of PI and HNE or DCI/HNE were analyzed by SDS-PAGE. Lane1, PI (1.5 µg); lane2, complex between PI (2.5 µg) and HNE (1.9 µg); lane3, complex between PI (2.5 µg) and DCI/HNE (1.9 µg); lane4, HNE (2.0 µg); lane5, size marker.



MALDI-TOF MS of PI-DCI/Trypsin and PI-Trypsin Complexes

Additional evidence for the presence of the COOH-terminal peptide in the denatured complexes was obtained by MS. Since the heterogeneous glycosylation of plasma PI, HNE, and cat G potentially could complicate data interpretation, we decided to concentrate on the non-glycosylated complexes between recombinant PI and trypsin. The masses of trypsin, PI, PI-DCI/trypsin, and PI-trypsin determined by MALDI-TOF are compared to the theoretical masses calculated from the primary structures (). The masses of trypsin and PI alone are in accordance with the expected intact molecules. The PI-DCI/trypsin and PI-trypsin complexes were stable during MS analysis, confirming the covalent nature of the complexes. SDS-PAGE of the HPLC-purified complexes (data not shown) revealed that free PI and PI-trypsin or PI-DCI/trypsin complexes coelute and are clearly separated from free trypsin. However, peaks corresponding to both PI and free trypsin or DCI/trypsin were observed during MS analysis, suggesting that some dissociation had occurred. This dissociation may be due to the much higher irradiation needed for desorption of the complexes. Dissociation due to the high irradiance also reduces mass accuracy as indicated in . However, the determined mass of 67.9 kDa for the PI-DCI/trypsin complex is consistent with a complex between intact PI and DCI/trypsin containing the COOH-terminal peptide. Prior to MS analysis the complex was analyzed by Edman degradation following SDS-PAGE and electrotransfer to PVDF membranes, confirming that the NH termini of both proteins were intact. The PI-trypsin complex subjected to MS was similarly analyzed by Edman degradation, revealing NH-terminal truncations of PI and trypsin at Lys-Thr in PI and Lys-Ser in the trypsin molecule. The determined mass of 53.0 kDa is in agreement with the theoretical molecular weight of this truncated PI-trypsin complex (). These observations support the SDS-PAGE data (Fig. 2) and suggest that denatured complexes between PI and active trypsin, like the complexes between PI and DCI/trypsin, contain the COOH terminus of PI.

Identification of the COOH-terminal Peptide Sequence in PI-Trypsin Complexes

PVDF membrane-bound samples were treated with CNBr to generate peptides from the COOH-terminal region in PI-trypsin and PI-DCI/trypsin complexes. Edman degradation of the generated fragments resulted in the release of several phenylthiohydantoin derivatives in the first two cycles. Before the third or fourth cycle, OPA was automatically applied to the sample (Fig. 3). This treatment blocks all primary NH groups but leaves secondary NH groups of Pro residues unaffected. Consequently, the number of phenylthiohydantoin derivatives acids was reduced, and from cycle 3 or 4 to cycle 10 we obtained the sequence (Pro)-Pro-Glu-Val-Lys-Phe-Asn-Lys. This sequence corresponds to P or P to P of the COOH-terminal region of PI and confirms that the region COOH-terminal to the reactive site is present in the SDS-stable PI-trypsin and PI-DCI/trypsin complexes.


Figure 3: Identification of the COOH-terminal sequence in PI-DCI/trypsin and PI-trypsin. Electroblotted complexes were cleaved with CNBr and subjected to 2 or 3 cycles of Edman degradation. NH-terminal residues other than Pro were blocked with OPA. Continued Edman degradation revealed the sequence of P or P to P of the COOH-terminal peptide. P indicates CNBr cleavage sites.




DISCUSSION

Much of our understanding of serine proteinase inhibitory mechanisms comes from x-ray analyses of the small standard mechanism inhibitors(19) . The standard mechanism of inhibition by proteins such as kunins and kazals can be envisioned as a lock and key interaction between the RSL and the substrate-binding sites of the enzyme. Covalent bonds are not involved, and the complexes are not stable to denaturing conditions because the interaction depends on the conformation of the involved proteins. Some attempts to understand the serpin mechanism of action have been made with the assumption that serpins fulfill the requirements of the standard mechanism(20, 21, 22) . As previously mentioned, serpin-proteinase complexes deviate from the standard mechanism by resisting dissociation in SDS-PAGE. The SDS-stable serpin-active proteinase complexes are believed to be stabilized by a covalent acyl-ester bond between the -carboxyl of the P residue and O of the proteinase Ser. This mechanism involves a denaturation-induced distortion of the catalytic triad whereby the COOH-terminal peptide is released and an acyl-ester bond is formed and trapped(1, 13, 14, 15, 23, 24) . This mechanism seems to be based primarily on a comparison to standard mechanism inhibitors and two observations; (i) the peptide COOH-terminal to the P residue is released following denaturation of the complex, and (ii) the formed complex is dissociated by nucleophiles such as hydrazine(25) .

Formation of complexes between serpins and inactivated proteinases have previously only been studied under native conditions(12, 25, 26, 27) . Here we show that serpin-inactive proteinase complexes including PI-DCI/HNE, PI-DCI/trypsin, and ACT-DCI/cat G resist dissociation when analyzed in reduced SDS-PAGE. This observation is incompatible with the scenario described above, since DCI inactivates serine proteinases by modifying the active site Ser(18, 28) . Consequently, the formation of an acyl ester bond involving O of Ser is not possible and other cross-links must account for the stability seen. Moreover, cleavage of the RSL P-P peptide bond cannot occur since the involved proteinases are catalytically inactive (Fig. 1). This was confirmed by the absence of free COOH-terminal peptides following analysis of the complexes by SDS-PAGE (Fig. 1, panels B, D, and F), mass spectrometry (), and Edman degradation (Fig. 3).

By comparing PI/HNE and PI-DCI/HNE in reduced SDS-PAGE, we noticed a small size difference (Fig. 3). This small size difference could not readily be explained by the expected loss of the 4.1-kDa COOH-terminal peptide from the PI/HNE complex. Edman degradation of the PI/HNE complex revealed that the NH terminus of PI had been truncated, releasing the peptide Glu-Thr. The loss of this 1.1-kDa peptide is consistent with the size difference noticed on SDS-PAGE (Fig. 2). This result suggested that the SDS-stable PI/HNE complexes contained the COOH-terminal peptide, as a size difference of 5.2 kDa would have been seen if this peptide was removed in addition to the NH-terminal peptide. To further investigate whether the COOH-terminal peptides were present in PI-DCI/trypsin and PI-trypsin, the complexes were purified and analyzed by MALDI-TOF MS () and Edman degradation. These analyses confirmed that the COOH-terminal peptide is present in complexes between serpins and active as well as inactive proteinases.

These results are at variance with previous hypotheses on serpin-proteinase interactions. These hypotheses were generated in part due to the appearance of COOH-terminal peptides in SDS-PAGE of complexes. Association of a serpin and a proteinase occasionally also results in the cleavage of a small amount of the serpin instead of forming a complex. This reaction might account for the occasional appearance of the free COOH-terminal peptide in SDS-PAGE.

These studies show that SDS-stable serpin-DCI/proteinase complexes are stabilized by covalent cross-links other than the acyl-ester bond involving Ser of the proteinase and the -carbonyl of the P residues. In addition, data from SDS-PAGE (Fig. 2), mass spectrometry (), and Edman degradation of electroblotted complexes indicate that SDS-stable complexes between serpins and active proteinases are stabilized by the same mechanism as serpin-DCI/proteinase complexes.

  
Table: Mass spectrometry of denatured PI-trypsin complexes

Complexes between PI and porcine trypsin were formed as described under ``Experimental Procedures'' and purified by reverse-phase HPLC. Complexes and controls were analyzed by MALDI-TOF mass spectrometry. The observed masses are compared to the calculated masses in the presence and absence of the COOH-terminal peptide of PI.



FOOTNOTES

*
This work was supported by National Institutes of Health NHLBI Grant HL49542. 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 Pathology, Duke University Medical Center, P. O. Box 3712, Durham, NC 27710. Tel.: 919-684-2872; Fax: 919-684-2920.

The abbreviations used are: RSL, reactive site loop; PI, -proteinase inhibitor; ACT, -antichymotrypsin; cat G, cathepsin G; DCI, 3,4-dichloroisocoumarin; DCI/HNE, 3,4-dichloroisocoumarin-inactivated HNE; DCI/trypsin, 3,4-dichloroisocoumarin-inactivated porcine trypsin; DCI/cat G, 3,4-dichloroisocoumarin-inactivated cathepsin G; DTT, dithiothreitol; HNE, human neutrophil elastase (EC 3.4.21.37); PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; MS, mass spectrometry; OPA, orthophthaldialdehyde; HPLC, high performance liquid chromatography.

The numbering used is the chymotrypsin numbering system.


ACKNOWLEDGEMENTS

We thank Drs. Tim Oury, Hanne Gr, and Eva H. Olsen for valuable suggestions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.