Biomedical Engineering Reference
In-Depth Information
II. ALPHA-1 ANTITRYPSIN
A.
Structure
The alpha-antitrypsin (AAT) gene is located on chromosome 14 (q31-32.3),
spanning 12.2 kb and consisting of seven exons and six introns (1). The cod-
ing exons (II-V) follow three exons (Ia, Ib, Ic), which code for the untrans-
lated region of the AAT gene (2,3). The gene is translated into a 418 amino
acid protein incorporating a 24 amino acid signal peptide. Glycosylation of
AAT occurs at three ASN sites—ASN 46, 83, and 247—in the endoplasmic
reticulum, and the protein is packaged in the Golgi apparatus prior to
release. The final 52-kDa protein is produced primarily in hepatocytes but
has also been shown to be expressed in epithelial cells, macrophages, and
neutrophils (4-6). Owing to the large number of AAT mRNA transcripts
present in the hepatocyte, it is believed that serum AAT is largely derived
from the liver from where it diffuses throughout the body and into the lung.
B. Activity
Alpha-antitrypsin is a member of the serpin family of serine protease inhi-
bitors (7). The tertiary structure of members of this family is similar as is
the exposed reactive center loop containing the inhibitory active site
sequence (8). Alpha-antitrypsin inhibits many proteases including trypsin,
plasmin, thrombin, factor X, and cathepsin G (4). However, the main cog-
nate protease of AAT is NE. Neutrophil elastase is a 29-kDa, 220 amino
acid protein synthesized in the promyelocytic stage of neutrophil develop-
ment (9). Neutrophil elastase is packaged into azurophilic granules from
which it can be rapidly released. Studies from NE knockout (-
=
-) mice have
revealed that the major function of NE is its microbicidal activity towards
gram-negative bacteria (10). Neutrophil elastase possesses an active site con-
taining a catalytic triad of residues—Ser173-His41-Asp88. Upon binding to
a protein substrate, molecular changes occur resulting in the transfer of an
electron to the Ser173 residue converting it to a reactive nucleophile that can
cleave a peptide bond in the target protein (11). However, when NE binds to
the active site of AAT, the Met358-Ser359 bond, cleavage of this Met-Ser
bond results in the generation of a tight, noncovalent interaction between
NE and AAT thereby inhibiting NE activity and release of the 36-amino
acid C-terminal Ser359-Lys394 peptide (12).
The oxidation susceptibility of AAT renders it less active against NE.
The association rate constant of AAT drops from 6
10
7
M
1
sec
1
to
3
10
4
M
1
sec
1
, a drop of almost 2000-fold (13). The molecular basis
of this oxidation was initially shown to be oxidation of the active site
Met358 residue (14). However, using recombinant-derived AAT, it has since
been demonstrated that Met351, another active site residue, is also involved in
the AAT-NE interaction (15). There are nine methionine residues present in
the AAT sequence; however, only Met 351 and 358 are fully surface exposed
