CONVENCIÓN MARCO DE NACIONES UNIDAS SOBRE CAMBIO CLIMÁTICO

Alpha]-antitrypsin is a member o f the serine protease inhibitor super-family o f which there are over 40 members (Kim and Yu, 1996). All members o f the serpin family are composed o f three p-sheets and several a-helices and are capable o f inhibiting, albeit to various degrees, those proteases which contain serine at their active site. The x-ray crystallographic structure o f ai-antitrypsin demonstrates that i t is m ade u p o f 3 p- sheets and 8 a-helices (Cox, 2001). The three-dimensional structure of ai-antitrypsin has been shown not to be the most thermodynamically stable folding intermediate possible. These modelling studies have also shown that ai-antitrypsin contains a highly stressed amino acid ‘loop’ at the C-terminal end o f the molecule, which contains the active site (Figure 1.25a). This loop has been demonstrated to be critical in maintaining the inhibitory function of the molecule.

The ai-antitrypsin present in the circulation is made up of 394 amino acids although a larger pre-protein containing a 24 amino acid hydrophobic signal sequence can be detected in the liver and in vitro. The protein sequence contains only one cysteine (residue 232) and therefore it does not have disulphide bonds (Figure 1.24a). However, the cysteine residue of ai-antitrypsin can form a disulphide bond with other proteins such as immunoglobulin a heavy chain, immunoglobulin k light chain and glutathione ( Cox, 2 001). It is a Iso known t o i nteract w ith h ydrophobic compounds such as cholesterol and bile acids (Janciauskiene and Eriksson, 1994). The cysteine residue of ai-antitrypsin has recently been shown to be extremely important in regulating and introducing other functions to the molecule when it becomes nitrosylated (Miyamoto et a l, 2000) (see Section 1.6.3.3).

Mature serum ai-antitrypsin is a globular glycoprotein o f approximate mass of 52 kDa with as much as 12% o f the molecular weight being contributed by carbohydrate (Cox, 2001). Alpha]-antitrypsin has three potential N-glycosylation sites at

C h apter 1.___________________________________Introduction

asparagine residues 46, 83 and 247, which are normally occupied by complex glycans giving ai-antitrypsin a net negative charge (Figure 1.25a). Isoelectric focusing of plasma -antitrypsin results in the detection of 8 bands, which are numbered M l to M8 (anodal-low pH to cathodal-high pH) (Fagerhol and Laurell, 1967). The M4 and M6 bands are the most abundant and these isoforms make up 40 and 34 percent of the total plasm a ai-antitrypsin, respectively (Cox, 2001) (Figure 1 .25b). T he multiple forms of a 1-antitrypsin are due primarily to the presence of different numbers o f sialic

acid residues on the glycans. Isoforms containing more triantennary complex glycans, and thus more sialic acid residues, have lower pi values. Bands M4 and M6 have one tri-antennary plus two bi-antennary glycans and three bi-antennary glycans, respectively (Vaughan et a l, 1982). The two minor cathodal iso forms, bands M7 and M8, have the same carbohydrate structure as the major bands, M4 and M6, but lack the five N-terminal amino acids (Glu-Asp-Pro-Gln-Gly) (Jeppsson et a l, 1985). The loss of these five amino acids, by post-translational cleavage, causes an additional cathodal shift o f the isoforms due to the loss o f the negatively charged amino acids, glutamic acid (Glu 1) and aspartic acid (Asp 2). The proposed structure o f the a r antitrypsin isoforms found in plasma is shown in Figure 1.25b. The genetic variation in the primary structure of ai-antitrypsin and its role in human disease are discussed as a preface to Chapter 5.

Figure 1.25. Met 358 Met 358 Asn 247 Asn 46 Asn 83 Front Back

o

•

e

O

Glycosylation Active site C ysteine Amino acid s involved site (Met358) (Cys232) in the reactive loop

(a)

, .Y V Y

Y V V

isV V

y

y Y

y

y y y

V

Y

amino acids amino acids _glycan glycan

1-394 6-394 _structure structure

(b)

Figure 1.25 (a) T h ree-dim ensional stru cture o f a , -antitrypsin sh ow ing the potential glycosylation sites, reactive center loop and the free-cystein e residue w hich can be readily nitrosylated to m odulate the function o f a ,-a n titr y p sin .

(b) E xplanation o f the heterogen eity o f plasm a a ,-a n titr y p sin observed during isoelectric focusing.

C h apter 1.___________________________________Introduction

1.6.3.3 Function and mechanism of action of ai-antitrypsin.

The main function of ai-antitrypsin is generally considered to be its protective role in the lower respiratory tract o f protecting lung endothelium from auto-proteolytic damage by neutrophil elastase. Any disturbance in this balance between protease and inhibitor due to a deficiency o f ai-antitrypsin can lead to destruction o f the components o f the lung extracellular matrix, especially elastin which provides elastic recoil in the connective tissue (Brantly et al., 1988), and an eventual fall in respiratory capacity o f the lung.

The active site of the ai-antitrypsin molecule is centred around the amino acid, methionine 358, and is situated in a highly stressed loop, which extends from leucine 327 to the C-terminus (Brantly, 1988) (Figure 1.25a). Oxidation o f methionine 358 destroys the inhibitory capacity towards neutrophil elastase. Methionine 358 is extremely susceptible to oxidation by environmental pollutants and patients with a i- antitrypsin deficiencies are advised to avoid smoking in order to not accelerate the onset o f emphysema (Brantly et a l, 1988). The serum levels o f oxidised ai- antitrypsin can be used as a marker of oxidative stress and have been shown to be elevated in conditions such as rheumatoid arthritis and in smokers (Ueda et a l, 2002). The mechanism by which neutrophil elastase is inhibited has been investigated by studying the docking o f the ai-antitrypsin molecule into the active site o f the protease (Carrell and Lomas, 2001). Neutrophil elastase is a protease secreted by neutrophils which cleaves peptide bonds at the carboxylic acid side o f methionine residues. The protease cleaves the -antitrypsin molecule between methionine 358 (active site) and serine 359 o f the highly stressed loop. This results in a large conformational change in ai-antitrypsin due to cleavage of the highly stressed loop which releases its energy like a coiled spring. Alphai-antitrypsin swings in a sling-shot motion to the side of the elastase and becomes tightly bound to the side of the protease, rendering it inactive (Carrell and Lomas, 2001). The inhibition reaction results in the suicide of both molecules and it has been calculated that it takes less than 0.2 seconds for one

C hapter 1.___________________________________Introduction

elastase (Brantly et a l, 1988).

Recently it has been shown that the free cysteine residue at residue 232 in the amino acid sequence can form disulphide bonds with other proteins and molecules such as albumin and glutathione (Miyamoto et a l, 2000). It has also been demonstrated that cysteine 232 can react with a nitrosonium ion to form a nitrothiol. Nitrosylated a i- antitrypsin ([NO]-ai-antitrypsin) still retains its inhibitory activity against neutrophil elastase but also has other properties (Miyamoto et a l, 2000). [NO]-ai-antitrypsin is a potent vasodilator of rabbit aortic rings in vitro in a dose-dependent manner and also has antimicrobial activity against both Gram-negative and Gram-positive bacteria. [NO]-ai-antitrypsin has a protective affect on the liver after hepatic reperfiision injury. Reperfusion of rat livers with [NO]-ai-antitrypsin in vivo after occlusion of the portal vein, was shown to have many beneficial features including decreased concentrations of liver enzymes in the plasma, increased hepatic blood flow, inhibition of neutrophil infiltration and decreased rates o f apoptosis (Ikebe et a l,

2000). Reperfusion with non-nitrosylated ai-antitrypsin did not produce these effects. Although the biochemical basis for these phenomena is unknown, it has been postulated that [NO]-ai-antitrypsin can act as a reservoir for nitric oxide, allowing delivery of NO to sites of action where it is required (Miyamoto et a l, 2000).

C hapter I. Introduction