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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 652-654, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.F250

Perspective

{alpha}1-Antitrypsin: Not Just an Antiprotease

Extending the Half-Life of a Natural Anti-Inflammatory Molecule by Conjugation with Polyethylene Glycol

Mark Brantly

Departments of Medicine, Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida

Address correspondence to: Mark Brantly, M.D., University of Florida College of Medicine, Division of Pulmonary and Critical Care Medicine, P.O. Box 100225 JHMHSC, Gainesville, FL 32610-0225. E-mail: brantml{at}medicine.ufl.edu

Abbreviations: {alpha}1-antitrypsin, {alpha}1AT • polyethylene glycol, PEG

Human {alpha}1-antitrypsin ({alpha}1AT), also called {alpha}1-proteinase inhibitor, is a mid-sized, bean-shaped glycoprotein secreted in abundance by hepatocytes, and by other tissues in lesser amounts (1). This especially diffusible protein is present in high concentrations in most tissues and is a major protein component of the lung epithelial lining fluid. Epithelial lining fluid levels of {alpha}1AT in nondeficient individuals are typically between 2 and 5 µM (2). During intense inflammatory events, such as pneumonia or acute respiratory distress syndrome, the lung is flooded with vast amounts of {alpha}1AT, and epithelial lining fluid levels can exceed 10 µM, sometimes approaching levels observed in the plasma (2–4).

Structurally, {alpha}1AT is a highly specialized bait for trapping proteases. The reactive site loop of {alpha}1AT extends several angstroms from its main structure. This loop is under substantial stress, much like a rubber rod tightly bent to form a horseshoe. Cleavage of the loop by neutrophil elastase is associated with relaxation of the loop and incorporation of much of the loop into one of the ß sheets of the molecule (Figure 1) (5–7). The newly formed carboxyl terminus partially inserts into the structure of neutrophil elastase, forming a covalent bond that typically does not disassociate in sodium dodecyl sulfate denaturing gels.



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  		Figure 1. Intact and neutrophil elastase–cleaved human {alpha}1-antitrypsin. Left, intact {alpha}1AT; right, cleaved {alpha}1AT. Residues in red, Met351 and the active site Met358; blue, Cys232; cyan, Ser359; black, reactive site loop; green, {alpha}-helices; yellow, ß-sheets. Images created using the open source molecular modeling program RasMol 2.6 (25). Coordinates for cleaved and intact {alpha}1AT were obtained from the Protein Data Bank (accession codes 9API and 1QLP).

 
{alpha}1AT has one cysteine and nine methionine residues. The sulfur groups of two of the methionines and the cysteine thiol group are potentially available for interaction with oxidants. Human {alpha}1AT has three N-linked complex carbohydrate side chains that are essential to its 4.5-d half-life in plasma (1). Yeast- or bacteria-produced recombinant {alpha}1AT have half-lives in plasma measured in minutes rather than in days, presumably because of their lack of mature carbohydrate side chains. However, when recombinant {alpha}1AT is delivered to the lung as an aerosol, its half-life is substantially longer (8). Clearance of {alpha}1AT in the plasma is likely similar to that of other secreted glycosylated proteins. {alpha}1AT clearance within the lung is less well understood, but at least a portion of {alpha}1AT is cleared by the SERPIN-enzyme complex receptor (9).

{alpha}1AT has a wide spectrum of antiprotease activity inhibiting several serine proteases found in the lung, including neutrophil elastase, protease 3, cathepsin G, plasminogen activator, and the lymphocyte granzymes. However, when rate of association is used as the defining factor to establish {alpha}1AT's major function, neutrophil elastase is clearly its primary protease target. Inhibition of neutrophil elastase is a primary function of {alpha}1AT (1). Although matrix proteins are neutrophil elastase's chief substrates, it clearly has activity against a wide variety of proteins, including cell surface receptors. Consistent with the observation, recent studies indicate that {alpha}1AT may be important in modulating neutrophil elastase cleavage of the phosphotidylserine receptor, a key receptor necessary for the recognition and disposal of apoptotic neutrophils (10–13). In this context, {alpha}1AT may play an important role in the resolution of inflammation.

In addition to its antiprotease activity, there is growing evidence that {alpha}1AT may have a broader function. {alpha}1AT blocks the cytotoxicity of neutrophil defensins, as well as their ability to upregulate epithelial cell production of interleukin-8 (14–16). Based on its methionine group content, it is plausible that {alpha}1AT may also act as an antioxidant. Although oxidation of the 358 and 351 methionine residues impairs the antiprotease activity of {alpha}1AT, the antioxidant activity may be an important biologic function separate from its role as an antiprotease (17–19).

There is now ample clinical evidence that in the absence of sufficient {alpha}1AT in the lower respiratory tract, there is an increased burden of proinflammatory factors (20, 21). Taken as a whole, {alpha}1AT appears to more than an antiprotease, and is better conceptualized as molecule with broad anti-inflammatory properties central to the regulation of neutrophil-mediated lung inflammation.

In this context, {alpha}1AT is a molecule well suited for engineering with the purpose of treating lung disorders characterized by a substantial component of inflammation. Re-engineering of {alpha}1AT was first accomplished in the 1980s, when variants of recombinant {alpha}1AT were created that varied the amino acids at the reactive site. These variants demonstrated that it is possible to broaden its antiprotease spectrum as well increase its resistance to oxidation, thus preserving antiprotease activity in the presence of an oxidant burden (22).

In this issue, Cantin and coworkers evaluate the biological consequence in the lung and plasma of conjugating {alpha}1AT with polyethylene glycol (PEG) (23). The conjugation of PEG to proteins, a process termed "pegylation," prolongs the half-life of proteins. Pegylation is a relatively new procedure initially demonstrated in the late 1970s and first used as a therapeutic application with the anticancer drug L-asparaginase (24). Subsequently, many other PEG-recombinant proteins have been introduced as therapies, including adenosine deaminase, interferon-{alpha}-2a, tumor necrosis factor, and interleukin-2. In addition to prolonging the half-life of these proteins, there is evidence that PEG conjugates may be less antigenic, further extending the potential usefulness of specific recombinant proteins (24).

Using a mouse model, Cantin and coworkers demonstrate that specifically conjugating 20 or 40 kD PEG to Cys232 extends the half-life of Escherichia coli–produced human {alpha}1AT in plasma and lung as compared with non–PEG-conjugated recombinant {alpha}1AT. Importantly, the authors demonstrate that PEG conjugation does not alter {alpha}1AT's rate of association with neutrophil elastase or its ability to form a sodium dodecyl sulfate–stable complex with neutrophil elastase. Finally, the authors demonstrate extended protection against lung injury induced by intratracheal instillation of human neutrophil elastase when the animals are treated with PEG-recombinant {alpha}1AT compared with non–PEG-recombinant {alpha}1AT (23). The authors' study does not explore the antigenicity of PEG-conjugated compared to free recombinant {alpha}1AT. Nor did they evaluate tissue distribution of PEG-conjugated {alpha}1AT compared with the nonconjugated form, or whether pegylation of {alpha}1AT might alter its non-antiprotease functions.

The implications of the study by Cantin and coworkers are several-fold. First, their observations pave the way for more extensive studies into the use of PEG-conjugated recombinant {alpha}1AT delivered by aerosol to the lung in neutrophil-mediated lung injury. In addition, because the half-life of recombinant {alpha}1AT can be extended, it is possible that the half-life of human plasma–purified {alpha}1AT may also be further extended by pegylation. Prolonging the half-life of the current form of {alpha}1AT used for treatment of {alpha}1AT-deficient individuals might help stretch limited supplies available worldwide by extending the dosage interval. In addition, increasing the half-life of recombinant {alpha}1AT in the plasma may provide an additional source of {alpha}1AT intravenous therapy for deficient individuals. Finally, the authors' study establishes an experimental template for aerosol delivery of other PEG-conjugated recombinant proteins to the lung.

In addition to recombinant {alpha}1AT, other recombinant proteins, such as granulocyte-monocyte colony-stimulating factor for pulmonary alveolar proteinosis and interferon-{gamma} for viral or mycobacterial infections, may be considered as PEG conjugates for aerosol delivery to the lung. Although the use of this approach in humans will need to evaluate the toxicity of PEG conjugates in the lung and explore issues involving potential compartmentalization of PEG-conjugated proteins, the study by Cantin and coworkers provides a scientific basis to explore new protein-based therapies for individuals with lung disease.


   Acknowledgments
 
This work was supported by grants from the Alpha-1 Foundation and the National Institutes of Health (HL00456).

Received in original form October 4, 2002


   References
Top
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Copyright © 2002 American Thoracic Society.