ÜBERSICHT Plasma-Kinin-Forming Cascade-Ac tivation
chain. Domain 4 consists of bradykinin and the
first 12 amino acids of the light chain.
120
1
00
Isolation of gC1qR and Cytokeratin 1 as HK-
Binding Proteins
In order to isolate binding proteins that interact
with HK, we prepared a soluble membrane pre-
paration from HUVECs and passed it over an HK
affinity column in the presence and absence of
8
6
4
2
0
0
0
0
0
5
0 μM zinc ion. We then eluted the column with
Factor XII
HK
dilute acid, neutralized the effluent, concentrated
it, and subjected it to SDS gel electrophoresis.
Three bands were observed in the presence of zinc
that were not visualized in its absence at 72 and 45
and 33 kD. The 33-kD band was the most pro-
minent and when we eluted the protein from the
gel and sequenced it, the first 13 amino acids were
identical to gC1qR, the receptor for the globular
heads of the C1q subcomponent of the first com-
ponent of complement (7). Employing cloned
gC1qR, we demonstrated zinc-dependent binding
to the light chain of HK but not to the heavy chain,
as well as zinc-dependent binding of Factor XII.
HK and Factor XII would compete for binding if
gC1qR is limiting (7). We also identified the 34-
Kd band to be gC1qR by immunoblot, employing
monoclonal antibody to gC1qR. Although C1q
binds to the N-Terminus of gC1qR, HK or Factor
XII binds to the C-Terminus, and HK binding can
be inhibited by a monoclonal antibody against
gC1qR. The binding site of the light chain of HK
has been mapped to residues 479–498 within do-
main 5 (3).
human IgG
0
10
100
1000
10000
[
Competitor], nM
Figure 1: High molecular
weight kininogen (HK)
competes with factor XII
for the same binding
Binding to Endothelial Cells
It has been shown that HK and Factor XII can each
bind to HUVEC (human umbilical vein endo-
thelial cells) in a reaction that is zinc dependent
sites on HUVEC. HUVEC
were incubated with 1
(
14, 6). Binding is saturable, reversible, and the
125
g/ml I-FXII in triplica-
number of binding sites in each protein is of the
order of 3x10 sites/cell with Kd of 40-50 nM for
HK and 12x10 sites/cell with Kd of 144 nM for
Factor XII (12, 13). Factor XII and HK can also
compete for binding, suggesting that they can in-
teract with the same cell surface macromolecules
6
te in the presence of in-
creasing concentrations
of unlabled factor XII
6
(
■), HK (●), or normal
human IgG (▲) for 120
min and bound ligand
determined. Percent bo-
und in the presence of a
competitor is plotted
against the concentrati-
on of the competitor.
(
Fig. 1). Prekallikrein does not have a receptor on
HUVEC, but is brought to the surface by virtue of
its ability to bind to domain 6 within the light
chain of HK (9). The HK domains that bind to
endothelial cells have been elucidated; this binding
involves heavy chain (domains 1–3) and light chain
In order to identify the binding site for HK
heavy chain, we again employed affinity chromato-
graphy, but rather than using HK or heavy chain as
the ligand, we employed a peptide that had been
mapped to domain 3 as the site reactive with endo-
thelial cells. This peptide was coupled to sepharose
and a cell membrane preparation passed over it in
the presence or absence of 50 FM zinc. After elu-
tion, zinc-dependent bands were found at 68 kD,
66 kD, and 33 kD. When biotinylated peptide was
used as a the ligand, it bound to the 68-kD band.
When we attempted to sequence this band, the N-
Terminus was blocked. We then digested it with
cyanogen bromide, separated the peptides by mass
spectroscopy, and sequenced a major peptide with a
mass of 2721. It proved to be an internal peptide of
the protein cytokeratin 1, in agreement with ob-
servations published by Hasan et. al. (4). Employ-
ing antisera to cytokeratin 1, we confirmed the
identity of the 68-kD band by immunoblot. We
also demonstrated HK binding to both cytokeratin
(
domains 5 and 6), with one site present on each
kDa
1
2
3
4
Figure 2: Analysis of the
proteins eluted from the
affinity columns using
biotinylated HK and
97 -
anti-cytokeratin 1. The
zinc-dependent binding
fractions were pooled
and subjected to a 10%
SDS/PAGE under redu-
cing conditions. After
electrophoresis, the pro-
teins were transferred to
nitrocellulose membra-
nes and were probed
with biotinylated HK
6
8 -
4
5 -
(
Lanes 1 and 2) and anti-
cytokeratin 1 (lanes 3
29 -
and 4). Lanes 1 and 3, the
eluate from LDC27 co-
lumn; lanes 2 and 4,
eluate from HK affinity
column.
1
and gC1qR (Fig. 2) by immunoblot, employing
the eluate from an HK affinity column and bio-
tinylated HK as ligand (8).
Allergo J 2000; 9: 407–10
408