Antibody Recognition of Chiral Surfaces
A R T I C L E S
ImmunoPure IgM purification kit no. 44897 was purchased from Pierce
(Rockford, IL). Secondary antibodies for enzyme-linked immunosorbent
assay (ELISA) were purchased from Jackson ImmunoResearch Inc.
(West-Grove, PA). The Vector-Red alkaline phosphatase substrate kit
was purchased from Vector Laboratories Inc. (Burlingame, CA). Color-
labeled crystals were viewed and photographed with a Nikon (Japan)
microscope equipped with a Lumina 2.2 CCD camera.
By definition, enantiomeric surfaces form diastereomeric com-
plexes with a specific antibody, and thus should be different
vis-a`-vis the antibody. Such examples at the molecular length
scale are abundant in biology and in antibody-antigen interac-
tions as well.16-18 The extent of chiral recognition at crystal
surfaces however depends heavily on the dissimmetry expressed
on the chiral surface,19,20 as well as on the resolution of the
interactions between the antibody and the surface. We have
recently raised and selected one antibody against crystals of
cholesterol monohydrate, which has very high affinity also for
monolayers of cholesterol at the air-water interface.21,22 This
antibody was shown to be highly stereoselective, but not
enantioselective, inasmuch as it recognized cholesterol and ent-
cholesterol (but not epicholesterol) monolayers with the same
high affinity.23 The absence of enantioselectivity was attributed
to the low level of dissimmetry expressed on the interacting
surface. Notwithstanding the fact that the surface is, by
definition, chiral, the close packing of the molecules prevents
molecular recognition of the hand-in-glove type. A surface-to-
surface recognition rather applies, which does not appear to be
influenced, in the case of cholesterol, by molecular chirality.
Here, we have studied molecular recognition of antibodies
on surfaces of crystals of the tripeptide L-leucine-L-leucine-L-
tyrosine.1,24 Enantiomorphism was used as a tool to test
recognition. Antibodies were selected against crystals of the LLL-
enantiomer, and their recognition was then characterized on both
enantiomeric crystals, LLL and DDD. The use of peptide crystals
introduces antigenic surfaces that may resemble, to some extent,
protein surfaces, nevertheless preserving the advantages of a
repetitive lattice whose structure is known at the atomic level.
A tripeptide is too short to be processed by the immune system
through the “classical pathway” 25 by which proteins and
polypeptides are processed. It is thus guaranteed that antibodies
will not be elicited against the single molecules, but rather
against arrays of molecular moieties exposed on the crystal
surface.
Synthesis of L-Leucine-L-Leucine-L-Tyrosine and D-Leucine-D-
Leucine-D-Tyrosine. N-t-Boc-Leu (6.7 g) and N-hydroxysuccinimide
(4.0 g) were dissolved in an ethyl acetate (100 mL)/dimethylformamide
(5 mL) mixture and cooled to 0 °C in an ice-water bath. Dicyclo-
hexylcarbodiimide (6.5 g), dissolved in ethyl acetate (30 mL), was
added. The reaction mixture was stirred for 1 h in an ice-water bath
and left overnight at room temperature. The dicyclohexylurea formed
was filtered, and the ethyl acetate was evaporated from the filtrate.
The activated N-t-Boc-Leu-succinimide was dissolved in dioxane (150
mL), and a solution of leucine (4.0 g) in 0.6 M NaHCO3 (150 mL)
was gradually mixed with the activated N-t-Boc-Leu-succinimide
solution. The reaction mixture was stirred overnight at room temperature
and then concentrated under vacuum. Water (100 mL) was added to
the original solution, which was then acidified with concentrated HCl
to pH 2. After 30 min in ice, the product N-t-Boc-Leu-Leu was filtered
and dried under vacuum. N-t-Boc-Leu-Leu was activated in a procedure
similar to the N-t-Boc-Leu activation, and dissolved in dioxane. Tyr-
(t-Bu) (3.5 g) was dissolved in a 0.6 M NaHCO3 (100 mL)/dioxane
(100 mL) mixture, and gradually mixed with the activated N-t-Boc-
Leu-Leu in dioxane solution. The reaction mixture was stirred overnight
at room temperature, and then concentrated under vacuum. The product
N-t-Boc-Leu-Leu-Tyr(t-Bu) was isolated by acidification as described
for N-t-Boc-Leu-Leu. The protecting groups were removed simulta-
neously by 2 h incubation in a mixture of 90% H2O, 5% trifluoroacetic
acid, and 5% triethylsilane (total 100 mL). The solvents were
evaporated, and the residue was triturated with ether (500 mL) and
dried under vacuum. Leu-Leu-Tyr was cleaned on a silica column,
eluted by a gradient of chloroform/methanol, 9:1 to 6:4. The final yield
was 5 g of Leu-Leu-Tyr (40 mol %). The whole procedure was
monitored by TLC in chloroform/methanol, 9:1, stained by potassium
permanganate. A commercial sample of L-leucine-L-leucine-L-tyrosine
was used as a reference. The product was identified by X-ray
crystallography, circular dichroism (CD), and NMR. The CD spectra
gave [θ]225 ) +26000 (deg cm2)/dmol and [θ]210 ) +20000 (deg cm2)/
dmol and [θ]225 ) -26000 (deg cm2)/dmol and [θ]210 ) -20000 (deg
cm2)/dmol for L-leucine-L-leucine-L-tyrosine and D-leucine-D-leucine-
Two antibodies were shown to interact with the chiral surfaces
at different levels of stereo- and enantioselectivity, implying
that they “feel” the same surface at a different resolution. One
antibody is stereoselective and enantioselective, while the other
has low stereoselectivity and no enantioselectivity. Understand-
ing this antibody specificity advances our understanding of
surface recognition in biological systems.
1
D-tyrosine, respectively. H NMR spectrum in CD3OH (Bruker 400
MHz): δ 0.83-0.89 (CH3 of leucines 1 and 2, 12H); 1.47-1.52 (CH,
CH2 of leucines 1 and 2, 6H); 2.8-3.1 (CH2 of tyrosine, 2H); 3.6 (CHR
of leucine1, 1H); 4.3-4.4 (CHR of leucine 2 and tyrosine, 2H); 6.8,
7.1 (aromatic H atoms of tyrosine, 4H).
Experimental Section
Crystallization of Leucine-Leucine-Tyrosine. Crystals of L-Leu-
L-Leu-L-Tyr for X-ray analysis were grown using the hanging drop
method from a 10 µL drop of a mixture of 80% water/20% 2-propanol
and a reservoir of 1 mL of a mixture of 90% water/10% 2-propanol.
The crystals grew within 48 h, at room temperature. Crystals of L-Leu-
L-Leu-L-Tyr for immunizations were grown in glass test tubes, from
mixtures of 70% water/30% 2-propanol, at room temperature. After
24 h, the mother liquor was removed and the crystals were suspended
(4 mg/mL) in phosphate buffered saline (PBS) saturated with Leu-
Leu-Tyr. Crystals of L-Leu-L-Leu-L-Tyr and D-Leu-D-Leu-D-Tyr for
ELISA were grown in bovine serum albumin (BSA)-coated polystyrene
24-well tissue culture dishes (Nunclon). The peptide was dissolved in
a mixture of 70% water/30% 2-propanol by heating (10 g/L). Upon
boiling the solution was removed from the hot plate and allowed to
cool for 5 min. Aliquots of 0.4 mL were placed in each well and seeded
by adding minute amounts of crushed crystals. The plate was covered
and left at room temperature for 48 h. The crystals grew, adhering to
the bottom of the well. The supernatant was then removed, and the
Materials and Equipment. Amino acids and protected amino acids
were purchased from Bachem Ag (Switzerland). Solvents and reagents
were purchased from Sigma-Aldrich Israel Ltd. (Rehovot, Israel).
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