290
Carbohydrate Antigens
interior residues are generally not accessi-
ble to such interactions. Carbohydrates are
built up by monosaccharides whereby the
enriched hydroxy groups readily interact
with water molecules by hydrogen bond-
ing. Their glycosidic linkages are more
flexible than the peptide bonds in pro-
teins and protein-like folding patterns are
not seen in polysaccharides. Thus, not
only are the terminals of the carbohydrate
chains accessible for molecular recogni-
tion but residues in the internal chain
are also exposed in solvent and are fre-
quently reactive.
This important concept was Frst estab-
lished in an immunochemical study focus-
ing on the Fne speciFcities of antidextran
antibodies. Immunochemical mapping of
the combining sites of two monoclonal
myeloma proteins speciFc for
α
(1
6) dex-
tran, W3129 and QUPC52, established
experimentally the existence of combin-
ing sites, which are speciFc for either
the internal linear chain or the terminal
structures of carbohydrate molecules (See
±ig. 6 for a schematic view of their binding
speciFcities). W3129 had a site saturated
by isomaltopentaose, whereas QUPC52 ac-
commodated isomaltohexaose. The bind-
ing constants were 1
×
10
5
M
1
for the
former and 5
×
10
3
M
1
for the latter.
±urther analyses showed the terminal
nonreducing glucose to contribute 50 to
60% of the total binding energy with
W3129 but less than 5% with QUPC52.
The two antibodies also differ in their abil-
ity to precipitate a synthetic linear dextran
with about 200 glucoses. QUPC52 but not
W3129 forms precipitins in saline. These
observations were interpreted to indicate
that the combining sites of QUPC52 must
be a ‘‘groove’’ into which internal chain
epitopes of
α
(1
6)linked glucose could
Ft; in contrast, the nonreducing ends of
dextran may be held by the cavity-type site
of W3129. Computer model building stud-
ies on two cavity-type mAb, W3129 and
16.4.12E, and one groove-type mAb 19.1.2
support the immunochemical distinction
of these two basic types of combining
sites. Recognition through either terminal
moieties or internal structures of a carbo-
hydrate molecule may also occur in other
protein–carbohydrate interactions.
X-ray crystallography visualizes the epi-
topes bound in their combining sites.
Such analyses have been performed on
several carbohydrate–antibody complexes.
The presence of terminal, branched ter-
minal, and internal chain epitopes of
polysaccharide antigens, as well as their
corresponding antibody-combining sites,
have been conFrmed. The crystal structure
ofthemurine±abS-20-4fromaprotective
anticholera, Ab speciFc for the lipopolysac-
charide Ag of the Ogawa serotype was
determined in its unliganded form and in
complex with synthetic fragments of the
Ogawa O-speciFc polysaccharide (O-SP).
The upstream terminal O-SP monosaccha-
ride was shown to be the primary antigenic
determinant to accommodate the antibody
binding pocket. This antibody-combining
site is immunochemically similar to a
cavity-type anti-
α
(1
6) dextran, W3129,
in that a single terminal sugar residue
accounts for 90% of the maximal bind-
ing energy. A
Salmonella
trisaccharide
epitope bound by mAb Se 155-4 was
also extensively studied. The ±ab of this
mAb was successfully cocrystallized with a
branched trisaccharide
α
D-Galp(1
2)[
α
-
D-Abep(1
3)]-
α
-D-Manp. The trisaccha-
ride Flls a hydrophobic pocket, 8-
˚
A deep
by 7-
˚
A wide, a size close to previous es-
timates of the minimum for anti-
α
(1
6)
dextran sites.
Unlike the above examples, whereby the
terminal or branched terminal epitopes
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