Philip A. Patston

Formation and properties of C1-inhibitor polymers

Heating of the serpin C1‐inhibitor above 55°C induced the formation of inactive polymers. Western blotting of non‐denaturing gels showed that the polymers bound to the conformation specific monoclonal antibody 4C3, suggesting that a similar conformational change to that occurring in complexed or cleaved inhibitor had taken place. N‐Terminal analysis of tryptic peptides which bound to 4C3 showed that the epitope resides within residues 288–444, a region which includes parts of β‐sheets A and C.α1‐Antichymotrypsin, α2‐antiplasmin, angiotensinogen and thyroxine binding globulin also polymerised on heating, indicating that this is a property of many serpins.

Significance of secondary structure predictions on the reactive center loop region of serpins: a model for the folding of serpins into a metastable state

To address how serpins might fold so as to adopt the mechanistically required metastable conformation we have compared the predicted secondary structures of the reactive center loops (RCLs) of a large number of serpins with those of the equivalent regions of other non‐serpin protein proteinase inhibitors. Whereas the RCLs of non‐serpin inhibitors are predicted to be loop or β‐strand, those of inhibitory serpins are strongly predicted to be α‐helical. However, non‐inhibitory serpins, which also adopt the metastable conformation, show no consistent preference for α‐helix. We propose that the RCL primary structure plays little role in promoting the metastable serpin conformation. Instead we hypothesize that preference for the metastable state results from the incorporation of part of the RCL into β‐sheet C, which as a consequence precludes incorporation of the RCL into β‐sheet A to give the most stable conformation. Consequently the RCL must be exposed and by default will adopt the most stable conformation in this particular context, which is likely to be an α‐helix irrespective of the primary structure. Thus the observed correlation between inhibitory properties in serpins and prediction of α‐helix in the RCL may instead reflect a need for alanine residues between positions P12 and P9 for functioning as an inhibitor rather than a structural or mechanistic requirement for α‐helix.

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds.

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.

Serine protease inhibitors (serpins).

Inhibition of serine proteases by serpins (serpin: serine protease inhibitor) is a key mechanism for the control of proteolysis in thrombosis, shock, and inflammation. The various members of the serpin gene superfamily (α(1)-antitrypsin, ovalbumin, C1-inhibitor, antithrombin III, α(2)-antiplasmin, type-1 plasminogen-activator inhibitor, and so forth) have many characteristics in common. In this article, we review the biochemistry and cell biology of serpins, and we discuss their clinical importance and therapeutic potential.

Serpin-ligand interactions

One of the more common features of serpins is the ability to bind various ligands. Ligand binding can occur so that the inhibitory properties of the serpin are regulated, so that the serpin can be localized, or to produce or modulate some other biological function of the serpin. Ligands known to affect serpin biologic activity include glycosaminoglycans such as heparin, heparan sulfate and dermatan sulfate, DNA, extracellular matrix proteins such as vitronectin and collagen, and small organic molecule hormones. Many different biochemical and biophysical techniques in conjunction with molecular biology and cell biology approaches have been used to study the binding of various ligands to serpins and to assess the influence of this binding on activity and structure. We summarize here the different approaches that have been used to identify serpin ligands and the many methods that have been used to characterize the interactions of these ligands with their cognate serpins.

Serpins and other serine protease inhibitors

In a recent Trends article, Salzet, Vieau and Stefano discuss the concept that serpins have evolved over a long period of time as part of a natural host defence mechanism1xSerpins: an evolutionarily conserved survival strategy. Salzet, M. et al. Immunol. Today. 1999; 20: 541–544Abstract | Full Text | Full Text PDF | PubMed | Scopus (36)See all References1. However, the authors confuse the meaning of the word ‘serpin’ as they mistakenly use it interchangeably with ‘serine protease inhibitor’. Although the original derivation of the word serpin was from serine protease inhibitor, serpins are just one of at least 23 distinct families of naturally occurring protein inhibitors of serine proteases2xWhat can structures of enzyme-inhibitor complexes tell us about the structures of enzyme-substrate complexes?. Laskowski, M. Jr. and Qasim, M.A. Biochim. Biophys. Acta Moll. Cell Res. 2000; 1477: 324–337PubMedSee all References2. Therefore, not all serine protease inhibitors are serpins.Of the serine protease inhibitors discussed only α1-antitrypsin (also called α1-proteinase inhibitor), α1-antichymotrypsin, α2-antiplasmin, antithrombin, C1-inhibitor, protease nexin I, and the Schistosome serpin, belong to the serpin family. As the authors correctly point out serpins have a core domain of about 350 amino acids in which there is much sequence homology between members. The serpins shown in Table 1 in their paper have been studied extensively, in part because their relatively high concentrations in plasma make them amenable to purification in large quantities, and because of the physiological and clinical significance of the proteolytic pathways they regulate. In addition, as is mentioned, some serpins, such as angiotensinogen and thyroxine binding globulin, do not inhibit proteases but have other functions. The properties of these and many other serpins have been reviewed extensively3xSerpins: Structure, function and biology. Gettins, P.G.W. et al. See all References3.The authors mention numerous other serine protease inhibitors, none of which are members of the serpin family. For example, aprotinin (also called bovine pancreatic trypsin inhibitor and Trasylol) and protease nexin II are both members of the Kunitz (BPTI) family. Much focus is given to protease inhibitors from invertebrates, although none of the proteins shown in Tables 3 and 4 are serpins. This is not to say however, that invertebrates do not produce serpins. It is becoming increasingly apparent that serpins play important roles in many invertebrates, such as Drosophila4xConstitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Levashina, E.A. et al. Science. 1999; 285: 1917–1919Crossref | PubMed | Scopus (322)See all References4 and Brugia malayi5xA novel serpin expressed by blood-borne microfilariae of the parasitic nematode Brugia malayi inhibits human neutrophil serine proteinases. Zang, X.X. et al. Blood. 1999; 94: 1418–1428PubMedSee all References5. Serpins are also critical to the life cycle of various pox-viruses. The understanding that viral serpins inhibit apoptosis has been very useful in determining the role played by caspases in this process6xSerpins and regulation of cell death. Bird, P.I. Results Probl. Cell Differ. 1998; 24: 63–89Crossref | PubMedSee all References6. The cowpox virus serpin crmA was found to inhibit interleukin 1-β converting enzyme, which is now called caspase-1. It is possible that mammalian intracellular serpins might also play a role in regulating apoptosis. In mammals the functions being ascribed to serpins are growing beyond their original description as serine protease inhibitors, with immune modulation7xEffects of endometrial serpin-like proteins on immune responses in sheep. Skopets, B. et al. Am. J. Reprod. Immunol. 1995; 33: 86–93Crossref | PubMedSee all References7, tumour suppression8xMaspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Zou, Z.Q. et al. Science. 1994; 263: 526–529Crossref | PubMedSee all References8, and angiogenesis inhibition9xAntiangiogenic activity of the cleaved conformation of the serpin antithrombin. O’Reilly, M.S. et al. Science. 1999; 285: 1926–1928Crossref | PubMed | Scopus (375)See all References9 being examples.Finally, Salzet et al. propose that serpins evolved earlier than had been thought, hence their abundance in invertebrates. Although in this context they are referring to serine protease inhibitors in general, it has already been suggested that the serpin family evolved at least 500 million years ago (see pages 4–10 of Gettins et al.3xSerpins: Structure, function and biology. Gettins, P.G.W. et al. See all References3 for the original references). An analysis of the nucleotide sequence homology between related rodent serpins, revealed that the region of the serpin which directly interacts with the serine proteinase and defines the target protease specificity of the serpin (the reactive centre loop) had diverged more than would be expected. It was suggested that such positive Darwinian evolution, resulted from a need for the serpins to adapt to newly encountered exogenous proteases from infectious agents or parasites10xAccelerated evolution in the reactive centre regions of serine protease inhibitors. Hill, R.E. and Hastie, N.D. Nature. 1987; 326: 96–99Crossref | PubMedSee all References10. One curious footnote to considerations of serpin evolution is that serpins have yet to be found in any bacteria or archaea. This could be indicative of when serpins first evolved or could merely represent that they have yet to be discovered.

Inhibition of plasma kallikrein by C1-inhibitor: role of endothelial cells and the amino-terminal domain of C1-inhibitor

Activation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1-inhibitor deficiency. Heterozygous individuals with <50% levels of active C1-inhibitor are susceptible to angioedema attacks indicating a critical need for C1-inhibitor to be present at maximum levels to prevent unwanted prekallikrein activation. Studies with purified proteins do not adequately explain this observation. Therefore to investigate why reduction of C1-inhibitor to levels seen in angioedema patients results in excessive kallikrein generation we examined the effect of endothelial cells on the inhibition of kallikrein by C1-inhibitor. Surprisingly, it was found that a C1-inhibitor concentration of greater than 1 microM was needed to inhibit 3 nM kallikrein. We propose that this apparent protection from inhibition was mediated by kallikrein binding to the cells via the heavy chain in a high molecular weight kininogen and zinc independent manner. Protection of kallikrein from inhibition was not observed when C1-inhibitor truncated in the amino-terminal domain by the StcE metalloproteinase was used, which suggests a novel function for this unique domain. The requirement for high concentrations of C1-inhibitor to fully inhibit kallikrein is consistent with the fact that reduced levels of C1-inhibitor result in the kallikrein activation seen in angioedema.

α1‐Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1

Coagulation and complement proteinases are activated in sepsis, and one approach to therapy is to develop proteinase inhibitors that will specifically inhibit these proteinases without inhibiting activated protein C, a proteinase that is beneficial to survival. In this study, we made mutants of the serpin α1‐PI, designed to mimic the specificity of C1‐inhibitor. The P3‐P2‐P1 residues of α1‐PI were changed from IPM to LGR and PFR, sequences preferred by C1s and kallikrein, respectively. Inhibition of C1s, kallikrein, factor XIIa, and activated protein C was assessed by SDS‐PAGE, and by determination of the kapp and SI. α1‐PI‐LGR inhibited C1s with a rate of 7790 M−1s−1, but only minimal inhibition of C1 in a hemolytic assay was observed. Kallikrein, factor XIIa, and activated protein C were inhibited with rates of 382,180 M−1s−1, 10,400 M−1s−1, and 3500 M−1s−1, respectively. α1‐PI‐PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4′ residue of α1‐PI‐LGR Pro to Glu reduced the activity with C1s, consistent with the idea that C1s requires hydrophobic residues in this region of the serpin for optimal interaction. The data provide insight into the requirements for kallikrein and C1s inhibition necessary for designing inhibitors with appropriate properties for further investigation as therapeutic agents.

Characterization of different high molecular weight angiotensinogen forms

BACKGROUND Angiotensinogen is the substrate for renin in the system that releases angiotensin II. This renin-angiotensin system is an important regulator of blood pressure (BP), and defects in the system are linked to the development of hypertension. Native angiotensinogen is a 62,000-dalton monomer, but various high molecular weight forms also exist, which have not been well characterized. High molecular weight angiotensinogen has been reported to be 5% of the total angiotensinogen, and increases to 60% of the total during pregnancy and hypertension. The purpose of this investigation was to study high molecular weight angiotensinogen in normal plasma. METHODS Normal human plasma was run on a gel filtration column, and high molecular weight angiotensinogen detected by Western blotting. Further purification was by ion-exchange chromatography. In vitro polymerization of angiotensinogen was analyzed by sodium dodecyl sulfate (SDS) and native gels. RESULTS Two forms of high molecular weight angiotensinogen were found with molecular weights of 500,000 and 250,000 daltons on gel filtration, and 140,000 and 110,000 daltons, respectively, on nonreduced SDS-polyacrylamide gel electrophoresis, and at 62,000 daltons on reduced gels. Our estimation of the amount of high molecular weight angiotensinogen present is close to that reported previously. We also describe some of the in vitro polymerization characteristics of angiotensinogen, which can be explained by angiotensinogen being a member of the serpin family of proteins. CONCLUSIONS The angiotensinogen polymers produced in vitro might provide a model system for some of the high molecular weight forms produced in vivo, and help in understanding their function.

Modulation of C1-Inhibitor and Plasma Kallikrein Activities by Type IV Collagen

The contact system of coagulation can be activated when in contact with biomaterials. As collagen is being tested in novel biomaterials in this study, we have investigated how type IV collagen affects plasma kallikrein and C1-inhibitor. Firstly, we showed C1-inhibitor binds to type IV collagen with a Kd of 0.86 μM. The effects of type IV collagen on plasma kallikrein, factor XIIa, and β-factor XIIa activity and on C1-inhibitor function were determined. Factor XIIa rapidly lost activity in the presence of type IV collagen, whereas plasma kallikrein and β-factor XIIa were more stable. The rate of inhibition of plasma kallikrein by C1-inhibitor was decreased by type IV collagen in a dose-dependent manner. These studies could be relevant to the properties of biomaterials, which contain collagen, and should be considered in the testing for biocompatibility.