Chemistry Reference
In-Depth Information
entist and writer (see the table and fi rst paragraphs in Chapter 15 for his promi-
nent role in the history of lectinology), we used washed red cells and performed
these assays in serum-free phosphate-buffered saline. Under these conditions, all
six
- defensins (RC - 1 - 3 and RTD -1-3) caused hemagglutination (Figure 26.4). The
addition of 30 mM cellobiose or lactose (for the structures of cellobiose/lactose,
please see Chapter 1) had no effect, but adding 30 mM chitobiose or 10% normal
human serum completely prevented hemagglutination. Chitobiose (see also
Chapter 18 for chitobiose-binding plant lectins) is a characteristic component of
chitin (see Chapter 12) which occurs in fungal cell walls, insect and arthropod
cuticles, and
N
-linked glycans. Among the other defensins, only rabbit
θ
α
- defensin
NP-1 (net charge +10), human
- defensins HBD -2 (net charge +5) and human
HBD-3 (net charge +13) caused hemagglutination. In later surface plasmon reso-
nance experiments, HBD-2 and -3 showed little or no ability to bind carbohydrates,
suggesting that hemagglutination resulted from their high net positive charge.
The jury remains undecided about NP-1, awaiting further experimental results.
To exclude the possibility that RC-1 acted through a charge- related mechanism,
we compared RC-1 with its
enantio
(RC - 112) and
inverso
(RC - 111) analogs, all of
which had a net charge of +4. RC-1 and -112 caused hemagglutination, but RC- 111
did not (Figure 26.5 ).
β
Figure 26.4
Hemagglutination by defensins.
Washed normal human erythrocytes in phos-
phate-buffered saline were mixed with 50
defensin subfamily (
α
,
β
or
θ
) is indicated
next to the row numbers.
Abbreviations:
RC, ret-
rocyclin; RTD, rhesus
θ
- defensin; HNPs and
HDs, human
α
- defensins; HBDs, human
β
- defensins; NP, rabbit
α
- defensin; RatNP, rat
α
-defensin. The rabbit and rat defensins were
purifi ed from leukocytes and the other defensin
peptides were synthetic.
g/ml
of defensin and allowed to settle. Agglutinated
red cells covered a large area at the bottom
of the microwell. Nonagglutinated red cells
formed a tight button at the center of the well,
as seen in the peptide-free control (arrow).The
μ
Although a red blood cell's surface looks smooth and featureless through a
regular microscope, it is covered by a dense thicket of glycoproteins called a gly-
cocalyx [4]. Glycophorin A (GpA), with over 1 million copies on each erythrocyte,
is among the most abundant molecules in this carbohydrate- rich surface coat.
GpA, a transmembrane protein that is around 60% carbohydrate by weight, is
bound by
θ
- defensins. RCs also self -associate and this property should allow them
