(Received for publication, December 4, 1995; and in revised form, January 10, 1996)
From the
Expression of the L-selectin adhesion molecule can be
rapidly down-modulated by regulated proteolysis at a membrane-proximal
site. The L-selectin secretase has remained undefined, and the
secretase activity is resistant to a broad panel of common protease
inhibitors. We have developed an L-selectin-alkaline
phosphatase reporter, consisting of the ectodomain of human placental
alkaline phosphatase fused to the membrane-proximal cleavage,
transmembrane, and cytoplasmic domains of L-selectin, to aid
in the screening for L-selectin secretase inhibitors. A
hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited
release of the L-selectin-alkaline phosphatase reporter in a
dose-dependent manner. The hydroxamic acid-based peptide was also found
to inhibit wild type L-selectin down-regulation from the
surfaces of phorbol myristate acetate-activated peripheral blood
lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of
the proteolytic cleavage fragments of L-selectin confirmed
that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with
formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting
that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin
secretase activity may involve a cell surface, zinc-dependent
metalloprotease, although L-selectin shedding is not affected
by EDTA and may be related to the recently described activity involved
in processing of membrane-bound TNF-.
The regulation of adhesion molecule expression and activity is an important facet of modulating cell adhesion and migration. Although a number of strategies to up-regulate receptor activity, both qualitatively and quantitatively, have been elucidated, mechanisms to rapidly down-regulate adhesion have been less well characterized. Expression of the L-selectin (CD62L) adhesion molecule can be rapidly down-regulated from leukocyte cell surfaces upon cell activation(1, 2, 3, 4, 5, 6, 7) . L-Selectin is cleaved at a membrane-proximal site, which releases a large soluble extracellular fragment(8) . Although the cleavage region is well conserved between human, mouse, and rat L-selectin, L-selectin proteolysis is remarkably resistant to the insertion of alanine point mutations at multiple positions surrounding the cleavage site(9, 10) . However short deletions in the cleavage region inhibit proteolysis, suggesting that the distance of the cleavage site from the membrane bilayer or the secondary structure of the cleavage domain is important for proteolysis(9, 10) .
A number of other leukocyte cell surface markers, including CD43, CD44, CD14, CD16, and CD50, have been shown to be down-regulated upon leukocyte activation(11, 12, 13, 14, 15, 16, 17) . However the down-regulation of L-selectin shows unusually rapid kinetics(1) . Moreover, CD14, CD16, CD43, CD44, and CD50 down-regulation is inhibited by a variety of serine protease inhibitors as well as metalloprotease inhibitors, such as EDTA and 1,10-phenanthroline(11, 12, 13, 14, 15, 16, 17) . In contrast, L-selectin proteolysis is resistant to these and a broad panel of other common protease inhibitors(11, 15, 18, 19) . Thus the class of protease involved in L-selectin shedding has remained unknown.
In this report, we have developed an enzyme
reporter construct consisting of the ectodomain of human placental
alkaline phosphatase fused to the cleavage, transmembrane, and
cytoplasmic domains of L-selectin to screen for novel
inhibitors of L-selectin proteolysis. We demonstrate that a
hydroxamic acid-based peptide, KD-IX-73-4 inhibits down-regulation of
the L-AP ()reporter. KD-IX-73-4 also inhibits
proteolysis of native L-selectin on peripheral blood
lymphocytes and PHA-stimulated lymphoblasts. This is the first compound
we have found that inhibits L-selectin proteolysis.
Figure 1:
Construction of an L-AP
reporter. A, schematic representation of the L-AP
reporter generated by fusing the 5` sequences encoding the signal
sequence and ectodomain of human placental alkaline phosphatase with
the 3` sequences encoding the cleavage, transmembrane, and cytoplasmic
domains of L-selectin. Cleavage of the L-AP chimera
by a putative membrane-bound protease (right-angle arrow) is
illustrated in the upper portion. The nucleotide and amino
acid sequences of the junction between amino acid 489 (glycine) of
alkaline phosphatase and amino acid 318 (lysine) of the L-selectin are shown in the lower portion. A single
serine residue is introduced at the junction. B, stable
expression of the L-AP reporter in the L1.2 pre-B cell line.
Unactivated (thick solid line) or PMA-stimulated (thin
solid line) L-AP transfectants were stained with primary
mAb directed against human placental alkaline phosphatase and a
phycoerythrin-conjugated second stage goat F(ab`) anti-mouse IgG and analyzed by flow cytometry. Background
staining with an isotype control primary mAb (R6.5) is also shown (dotted line). C, immunoprecipitation of the L-AP reporter. Two L-AP-transfected L1.2 cell lines (L-AP1.5 and L-AP3.2) and mock transfectants were
metabolically labeled with [
S]methionine and
[
S]cysteine. Cell lysates were
immunoprecipitated with an antibody directed against the ectodomain of
alkaline phosphatase (lanes 5 and 6) or with
antiserum directed against the cytoplasmic domain of L-selectin (lanes 2 and 3). The membrane
bound L-AP chimera is indicated with the left arrow.
The released soluble human placental alkaline phosphatase was
immunoprecipitated from cell-free supernatants of labeled cells with an
anti-human placental alkaline phosphatase antibody (lanes 8 and 9). D, assay for released alkaline
phosphatase activity. Cell-free supernatants were collected from
unactivated and PMA-stimulated parent L1.2 cells and two L-AP-transfected lines, L-AP1.5 and L-AP3.2.
An aliquot of the supernatant was mixed with a chemiluminescent
substrate for alkaline phosphatase, as described under ``Materials
and Methods.'' Emitted light was detected and quantitated on a
luminometer. Exogenously added alkaline phosphatase is shown as a
positive control. Mean values of triplicate samples (± S.D.) for
one experiment are shown and are representative of eight similar
experiments.
Alkaline phosphatase activity was initially assessed in
adherent COS transfectants by the addition of naphthol phosphate and
Fast Red chromagen (Biogenex, San Ramon, CA), which formed a red
precipitate around L-AP-transfected cells. The
activation-dependent release of the alkaline phosphatase reporter into
the supernatant was assessed by harvesting cell-free supernatants from
unstimulated or PMA-stimulated L-AP transfectants. L-AP transfectants (2 10
cells/ml) were
preincubated with compounds for 10 min at room temperature and then
activated with 100 ng/ml PMA for 30 min at 37 °C. Cells were
pelleted, and 100 µl of cell-free supernatant was harvested and
analyzed for released alkaline phosphatase activity using the Phospha
Light Reporter Assay (Tropix, Bedford, MA). Endogenous alkaline
phosphatase activity was inactivated by heat treatment for 30 min at 65
°C. The heat-resistant placental alkaline phosphatase was measured
by the addition of reaction buffer and CSPD chemiluminescent substrate
as per the manufacturer's instructions (Tropix, Bedford, MA).
Chemiluminescence was quantitated on a luminometer (Berthold AutoLumat
LB953). Samples were performed in duplicate or triplicate, as
indicated.
Preparation of
HONHCOCHCH(CH
CH(CH
)
)CO-Nal-Ala-NH
.
To the chilled (0 °C) solution of above t-butyl ester
(0.512 g) and L-napthylalanyl-L-alanine
amide hydrochloride (0.81 g) in dimethylformamide was added
triethylamine (0.42 ml) and then slowly by parts N-ethyl-N`-(3-dimethylaminopropyl) carbodiimide (0.48
g) over 15 min. The reaction mixture was stirred for 2 h at 0 °C
and left overnight at room temperature. The next day, ethyl acetate
(100 ml) was added, and the solution was washed 3 times with 50-ml
portions of 1 N HCl, NaHCO
(sat), brine and then
dried over magnesium sulfate. A white solid (0.44 g) obtained after
evaporation of solvent was treated with 6N HCl/dioxane at room
temperature for 1 h to remove the t-butyl ester group.
Evaporation of dioxane and precipitation with ethyl ether gave a white
solid (0.32 g) that was then coupled to O-benzyl
hydrolyamine using the N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide
procedure with triethylamine. Catalytic hydrogenation (10% Pd/C, MeOH,
4 h) furnished the hydroxamic acid product as a mixture of two
diastereomers. The isomers were isolated by reversed phase
chromatography on a C-18 Dynamax column using an isocratic TFA/AcCN
elution mixture. The initial products were lyophilized and
characterized as specified below. NMR (Isomer I): (300 MHz,
Me
SO - d
);
(ppm) 0.64 and 0.73 (6H, 2xd, (CH
)
CH); 0.90 (3H, m,
(CH
)
-CH-CH
; 1.22 (3H, d, CH
-CH); 2.64 and 3.00 (1H and 1H, m, CH
-C
H
); 1.84-1.96
(3H, m, CO-CH
-CH); 4.20 (1H, m, CH-CH
CH
); 6.984 and 7.21 (1H and 1H,
s, CO-NH
); 7.40-7.90 (8H, m, C
H
and
CH
-CH-CO-NH
); 8.18 (1H, d,
CO-NH-CH-CH
); 8.66 (1H, s, NH-OH). FABMS:
calc. (M+H) 457.23, found 457. HPLC (Isomer I): Rt = 13.5
min, Hibar C
(4.7
150 mm), gradient 30-60% B
in 30 min, A: 0.05% TFA; B: 0.05% TFA in AcCN, 1 ml/min. NMR (Isomer
II): (300 MHz, Me
SO - d
);
(ppm) 0.24 (3H,
d, (CH
)
CH); 0.38 (4H, m, (CH
)
CH and
(CH
)
CH); 0.70 and 1.08 (1H and 1H, m,
(CH
)
-CH-CH
); 1.34 (3H, d, CH
-CH); 1.92 and 2.02 (1H and 1H, s,
CO-CH
-CH); 2.86 and 3.34 (1H and 1H, m, CH
-C
H
); 2.47 (1H, m,
CH
-CH-CO); 4.23 (1H, m,
CH-C
H
); 4.55 (1H, m,
CHCH
); 7.02 and 7.15 (1H and 1H, s,
CO-NH
); 7.44-7.78 (7H, m,
C
H
); 8.00 (1H, d,
CH
CO-NH-); 8.42 (1H, d,
CO-NH-CH-CH
); 8.68 (1H, s, NH-OH); 10.50
(1H, s, NH-OH). FABMS: calc. (M+H) 457.23, found 457.
HPLC (Isomer II): Rt = 15.9 min, Hibar C
(4.7
150 mm), gradient 30-60% B in 30 min, A: 0.05% TFA; B:
0.05% TFA in AcCN, 1 ml/min.
Alkaline phosphatase activity of the L-AP reporter was confirmed by reaction of COS cell transfectants with naphthol phosphate and Fast Red chromagen, a substrate that reacts with alkaline phosphatase and resulted in the formation of a red precipitate around L-AP-transfected cells but not around mock-transfected cells (data not shown). The PMA-induced cleavage of the reporter construct was assessed by harvesting cell-free supernatant from L-AP stable transfectants and assaying for released alkaline phosphatase activity with a chemiluminescent substrate (Fig. 1D). The L-AP transfectants showed a low background of alkaline phosphatase release in the absence of PMA activation. However upon stimulation with PMA, a 6-8-fold increase in alkaline phosphatase activity was released into the supernatant. No alkaline phosphatase activity was detectable from the parent L1.2 cells in the presence or absence of PMA (Fig. 1D).
Figure 2: Release of the L-AP reporter is inhibited by hydroxamic-based peptides. A, structure of KD-IX-73-4 (N-{L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl}-L-3-(2`-napthyl)alanylL-ala-alanine amide) and its diastereomer KD-IX-73-5 (N-{D-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl}-L-3-(2`-napthyl)alanyl-L-alanine amide). The diastereomers were separated by reverse phase HPLC. B, inhibition of L-AP reporter release. L-AP transfectants were preincubated with various concentrations of compounds as indicated for 10 min and then activated with 100 ng/ml PMA for 30 min at 37 °C. Cell-free supernatants were harvested and assayed for released alkaline phosphatase activity as described in the legend to Fig. 1. Mean values (± S.D.) of duplicate samples for one experiment are shown and are representative of four similar experiments.
Figure 3: Hydroxamic acid compounds inhibit L-selectin down-regulation on lymphocytes. Peripheral blood lymphocytes were treated with KD-IX-73-4 or its diastereomer KD-IX-73-5, as indicated, and activated with 100 ng/ml PMA for 30 min at 37 °C as indicated. Some cells, as indicated, were pretreated with 50 µg/ml KD-IX-73-4 and then washed twice to remove compound prior to the addition of PMA. The cells were stained with phycoerythrin-conjugated Leu-8 anti-L-selectin mAb or with phycoerythrin-conjugated control mAb (not shown), and 10,000 events per sample were analyzed by quantitative flow cytometry. Mean fluorescence values (± S.D.) from three experiments are plotted.
To confirm that the KD-IX-73-4 inhibited L-selectin proteolysis, cleavage products of L-selectin were analyzed by immunoprecipitation from metabolically labeled PHA-stimulated lymphoblasts. We have previously shown that the 74-kDa membrane form of L-selectin is cleaved to release a 68-kDa soluble fragment and a 6-kDa transmembrane fragment(8) . Immunoprecipitation from cell lysates with JK564 serum directed against the cytoplasmic domain of L-selectin revealed that treatment with KD-IX-73-4 prevented the disappearance of the full-length 74 kDa L-selectin and inhibited appearance of the 6-kDa transmembrane cleavage product from PHA lymphoblasts stimulated with PMA (Fig. 4, lane 3 versus lane 2). Similarly immunoprecipitation from cell-free supernatants with JK923 serum directed against the ectodomain of L-selectin revealed that treatment with KD-IX-73-4 inhibited the release of the 68-kDa soluble form of L-selectin (Fig. 4, lane 6 versus lane 5). The small amount of cleavage products observed in the KD-IX-73-4-treated lymphoblasts may be due in part to spontaneous proteolysis that occurred during the metabolic pulse and chase periods, when the KD-IX-73-4 compound was not present.
Figure 4:
Hydroxamic acids inhibit proteolysis of L-selectin. Day 5 PHA lymphoblasts were metabolically
pulse-labeled with [S]methionine and
[
S]cysteine for 30 min and chased for 30 min in
the absence of inhibitors. Labeled cells were then preincubated with
KD-IX-73-4 compound for 10 min as indicated and then activated with 100
ng/ml PMA for 30 min as indicated. Cell lysates were immunoprecipitated
with JK564 serum directed against the cytoplasmic domain of L-selectin. Cell-free supernatants were immunoprecipitated
with JK923 serum directed against the ectodomain of L-selectin. Samples were subjected to SDS-polyacrylamide gel
electrophoresis and autoradiography with
fluorography.
Figure 5:
L-Selectin is cleaved in cis.
Neutrophils and lymphocytes were co-cultured at a ratio of 7:1 and a
total cell concentration of 1 10
cell/ml. The cell
mix was left unstimulated or was stimulated with 50 ng/ml PMA or
10
M fMLP, as indicated, for 30 min at 37
°C. The cells were stained with phycoerythrin-conjugated Leu-8
anti-L-selectin mAb or with phycoerythrin-conjugated control
mAb (not shown) and analyzed by quantitative flow cytometry. L-Selectin expression on neutrophil versus lymphocyte
populations was determined by appropriate forward- and side-scatter
analysis gates. Mean fluorescent values (± S.D.) for two
separate experiments are plotted.
We have found that L-selectin proteolysis is inhibited by a hydroxamic acid-based compound, KD-IX-73-4. Mechanistically the hydroxamic acid moiety functions to coordinate zinc metal, and thus the hydroxamic acid-based peptides are potent inhibitors of zinc-dependent matrix metalloproteases such as collagenase and gelatinase(20) . Our results suggest that the L-selectin secretase may involve a metal-dependent protease, possibly a member of the metalloprotease family. However, previous results have shown that L-selectin down-regulation is resistant to several general metalloprotease inhibitors, such as 1,10-phenanthroline (11) and EDTA (11, 15, 18, 19) . In contrast, matrix metalloproteases are inhibited by both EDTA and 1,10-phenanthroline. Thus the exact class of protease cannot be ascertained at this time. The observation that EDTA but not KD-IX-73-4 inhibits the metal-dependent activity of exogenous alkaline phosphatase suggests that KD-IX-73-4 is not acting nonspecifically as a general metal chelator.
Although the KD-IX-73-4 compound is a potent inhibitor of collagenase and gelatinase, it is unlikely that the natural L-selectin secretase is the common soluble form of collagenase. The potency of KD-IX-73-4 against L-selectin secretase is about 1000-fold less than that for collagenase(20) . Moreover two specific inhibitors of collagenase and gelatinase were previously found to be inactive in inhibiting L-selectin proteolysis(11) . Our indirect evidence suggests that the L-selectin secretase is a membrane-bound activity. Neutrophils and lymphocytes both respond to PMA activation and down-regulate L-selectin from their cell surfaces. However, fMLP stimulates only neutrophils to down-regulate L-selectin, even if lymphocytes are co-incubated with fMLP-stimulated neutrophils at high cell densities. These results suggest that the secretase acts in cis with the membrane-bound L-selectin. A membrane-bound protease constrained in the same two-dimensional membrane plane as L-selectin would effectively increase the local substrate concentration for the putative secretase and may explain in part the rapid kinetics of L-selectin proteolysis. Indeed the relatively poor potency of the KD-IX-73-4 compound may be due in part to the rapid kinetics of the secretase activity following cell activation.
Recently three groups have shown
that hydroxamic acid-based peptides can inhibit processing of a
membrane-bound form of TNF- to a soluble
form(21, 22, 23) . KD-IX-73-4 is structurally
identical to compound one in the study by Mohler et
al.(21) , and its potency against TNF-
processing is
similar to what we found for L-selectin proteolysis. A similar
compound, TNF-
protease inhibitor, has also been found to inhibit
release of the TNF receptor(24) . Notably TNF-
processing,
in contrast to L-selectin proteolysis, is inhibited by
EDTA(21) . Moreover TNF-
is a type II protein, whereas L-selectin is a type I protein. L-Selectin and
TNF-
are among an emerging class of diverse proteins, such as
transforming growth factor-
(25, 26, 27) ,
-amyloid precursor
protein(28, 29) , IL-6
receptor(30, 31) , TNF receptor(32) , and
angiotensin-converting enzyme(33, 34) , which are
regulated by rapid and inducible cleavage from the cell
surface(35) . Significantly, the native protease involved in
any of these secretase activities has not been described. The
availability of a protease inhibitor will aid in the purification and
identification of the natural L-selectin secretase.
Finally, the hydroxamic acid-based inhibitors will allow us to probe
the contribution of L-selectin shedding to L-selectin
function. Recently we have demonstrated that hydroxamic acid-based
inhibitors significantly reduce neutrophil rolling velocity on L-selectin ligands in an in vitro model of
hydrodynamic flow. ()These results suggest that L-selectin proteolysis occurs during L-selectin-mediated rolling interactions and that shedding
contributes significantly to the velocity of rolling. The multistep
adhesion cascade model predicts a well orchestrated interplay of
selectin, chemokine, and integrin functions. It is possible that L-selectin shedding facilitates this transition and that
inhibition of L-selectin shedding might impact downstream
events, including transendothelial migration.