Biomedical Engineering Reference
In-Depth Information
18.2 ANTITHROMBIN
18.2.1 C
HEMICAL
S
TRUCTURE
OF
A
NTITHROMBIN
AT is a glycosylated polypeptide that belongs to a large family of serpins that have common
structural and functional homology.
50-52
Over the entire group of these serpin proteins, the pri-
mary sequence homology is approximately 30%, and several tertiary structure characteristics
are shared.
53
AT is synthesized in the liver and its transcript comes from a gene present on the
long arm of chromosome 1.
54
In humans, the AT gene is spread over a continuous 19 kb stretch
containing seven exons and six introns,
55,56
with an open reading frame of 1396 nucleotides that
includes a 96-nucleotide-stretch coding for a 32-amino acid-signal peptide (which is removed from
the N-terminal of AT prior to cellular secretion).
56
In the AT sequence, mammalians have sequence
variation of 10-15% but high conservation around the reactive site.
57
There are several important aspects of AT primary structure that create impact on its function
and play a role in the more native covalent complexes prepared from heparin. This plasma serpin is
a 60,000 Da single-chain polypeptide
58-60
that contains 432 amino acids and three pairs of disulfi de
bonds.
61
In particular, two of the disulfi des link a somewhat unstructured length of 45 amino acid
residues at the N-terminus with the third and fourth α-helices in AT. As will be seen later, the short
N-terminal sequence beyond the last disulfi de-bonded cysteine is an important feature involved in
ATH formation from UFH. In terms of tertiary structure, AT from human plasma has 31% α-helix,
16% β-sheet, 9% β-turn, and 44% random coil.
62,63
Considering the type of the amino acid comple-
ment, AT has a net neutral p
I
because of the presence of signifi cant amount of arginyl and lysyl
residues that are the main contributors to the high-affi nity UFH-binding regions.
53
A number of
attempts have been made to get a full description of the AT three-dimensional structure to under-
stand its functional activity. To this end, an x-ray crystal structure of the serpin with 3 Å resolution
has been produced.
64
This analysis and studies by others have shown that AT can exist in either an
active or an inactive (or latent) form
65
that occur together as a dimer during crystallization. Models
of the active structure show a glycoprotein inhibitor with nine α-helices and three β-sheets.
64
Posttranslational modifi cation is restricted to complex type asparaginyl N-linked glycosylation
that plays an important role in the functional activity of AT with regard to UFH catalysis of the
inhibitor's reactions.
In vivo
AT glycosylation is carried out by a glycosyl-transferase at asparagines
that are accompanied by specifi c neighboring sequences in areas of β-sheet structure. The ini-
tial step in this process involves placement onto the asparaginyl R-group of a tetraantennary,
high-mannose glycan by transfer from a dolicol phosphate. These high-mannose glycans are then
degraded by endoglycosidases to give a chitobiose-trimannose core, which acts as a base for the
addition of monosaccharide residues to form the carbohydrate structures observed in the fi nal gly-
coprotein that is excreted.
66
During this processing, variation can occur in the fi nal type of glycan
structures produced. For example, different degrees of branching are observed radiating out from
the glycan core. Previous work has shown that glycosylation sites on AT can display mono-, bi-,
tri- and tetraantennary-branched oligosaccharide chains.
67,68
In addition to heterogeneity in branch-
ing, the degree of substitution of terminal
N
-acetylneuraminic acid residues on AT glycans also
varies.
69
Further variation is evident with the addition of fucosyl residues on glycans at Asn96
and Asn192.
67
In addition to alteration in the glycan structures themselves, subforms of AT have
been described in which the number of oligosaccharides per polypeptide chain differs. AT mol-
ecules with three,
70
four,
71
and even fi ve glycans have been discovered.
72
A number of factors have
been muted as critical for infl uencing the degree of AT glycosylation. One general condition called
carbohydrate-defi cient glycoprotein syndrome leads to decreased glycosylation of AT by the golgi.
73
These under-glycosylated AT derivatives exhibit lower activity in anticoagulant functional tests.
Overall, the main glycoforms of mammalian AT contain either four glycans (α-AT) or three glycans
(β-AT) per molecule. Thus, in human plasma-derived AT, the fully glycosylated α-AT has N-amido
glycosidically linked glycans on asparaginyl residues at positions 96, 135, 155, and 192.
74
The β-AT
