©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Shedding of the Lymphocyte

L

-Selectin Adhesion Molecule Is Inhibited by a Hydroxamic Acid-based Protease Inhibitor

IDENTIFICATION WITH AN L-SELECTIN-ALKALINE PHOSPHATASE REPORTER (*)

(Received for publication, December 4, 1995; and in revised form, January 10, 1996)

Carol Feehan Krzysztof Darlak (1) Julius Kahn Bruce Walcheck Arno F. Spatola (1) Takashi Kei Kishimoto (§)

From the Department of Immunological Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877 and the Departments of Chemistry and Biochemistry, University of Louisville, Louisville, Kentucky 40292

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-alpha.


INTRODUCTION

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 (^1)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.


MATERIALS AND METHODS

Antibodies

DREG-200 mAb (IgG1) directed against the ectodomain of L-selectin (7) and CA21 mAb (IgG1) and polyclonal antiserum JK564 directed against the cytoplasmic domain of L-selectin (8) have been previously described. Phycoerythrin-conjugated Leu-8 (anti-L-selectin) and isotype control mAb were purchased from Becton-Dickinson (San Jose, CA). Monoclonal antibody 8B6 and polyclonal serum directed against human placental alkaline phosphatase were purchased from Dako (Glostrup, Denmark).

Peripheral Blood Leukocytes

Peripheral blood mononuclear leukocytes were isolated from normal healthy volunteers as described previously(8) . PHA lymphoblasts were generated by stimulation of mononuclear leukocytes with 2.5 µg/ml PHA for 5 days as described previously(8) .

L-AP Reporter

The pCMV/SEAP expression vector encoding a soluble form of the human placental alkaline phosphatase gene was purchased from Tropix (Bedford, MA). The L-selectin cDNA in the CDM8 vector was a generous gift of Dr. Brian Seed. The pCMV/SEAP vector was cleaved with HpaI and XbaI to remove the stop codon and 3`-untranslated sequences of the alkaline phosphatase gene. A fragment encoding the cleavage region (starting with lysine at position 318), transmembrane domain, cytoplasmic domain, and 3`-untranslated regions of L-selectin was amplified by polymerase chain reaction with a specific 5` L-selectin primer (5`-CCAAATTGGACAAAAGTTTC-3`) and a 3` CDM8 universal primer (5`-CCACAGAAGTAAGGTTCCTTCACAAAG-3`). The 3` end of the amplified fragment was cleaved with XbaI, and the fragment was ligated in-frame with the HpaI- and XbaI-digested pCMV/SEAP vector, introducing a single serine residue at the boundary of the alkaline phosphatase and L-selectin sequences (see Fig. 1A). The chimeric gene was confirmed by DNA sequencing and expressed transiently in COS cells or stably in L1.2 pre-B cells co-transfected with a puromycin selectable marker. Stable lines were initially selected in 3 µg/ml puromycin and then analyzed for cell surface expression of human placental alkaline phosphatase.


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`)(2) 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 times 10^7 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.

Metabolic Labeling

L-AP transfectants and day 5 PHA lymphoblasts were metabolically labeled with [S]methionine and [S]cysteine as described previously(8, 9) .

Immunoprecipitation

Cell lysates and cell-free supernatants were immunoprecipitated as described previously(8, 9) . Immunoprecipitated samples were run on tricine-SDS-polyacrylamide 10-20% gradient gels (Novex). Gels were then fixed in 30% methanol, 10% acetic acid, 1% glycerol, treated with an autoradiography enhancer (Entensify A/B, DuPont NEN), dried in cellophane, and exposed to x-ray film (Kodak X-OMAT) at -70 °C.

Western Blot Analysis

Western blot analysis with the CA21 mAb was performed as described previously(8) , with the exception that the blot was visualized by the addition of ECL chemiluminescent substrate and exposure to film as per the manufacturer's instructions (Amersham Corp.).

Fluorescence-activated Cell Sorter Analysis

Cells were stained directly with 5 µl of phycoerythrin-conjugated Leu-8 anti-L-selectin mAb or isotype control mAb (Becton Dickinson) or indirectly with 8B6 anti-alkaline phosphatase mAb or isotype control mAb followed by a second stage phycoerythrin-conjugated goat F(ab`)(2) anti-mouse IgG serum in phosphate-buffered saline containing 5% goat serum at 4 °C for 30 min. Cells were then washed twice with goat serum/phosphate-buffered saline fixed in 1% paraformaldehyde/RPMI medium, and 10,000 cells/sample were analyzed by flow cytometry on a FACScan machine (Becton Dickinson).

Synthesis of L-Napthylalanyl-L-alanine Amide Hydrochloride (L-Nal-L-Ala-NH(2))

Boc-L-Nal-L-Ala-NH(2) was prepared in 90% yield from Boc-Nal-OH and Ala-NH(2) by N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide activation. The HCl salt of the compound was obtained by treatment of the Boc-Nal-Ala-NH(2) product with 10 N HCl/dioxane, followed by removal of solvent, drying, and crystallization from methanol.

Synthesis of 3-Ethoxycarbonyl-3-(2-methylpropyl)propanoic Acid

Potassium t-butoxide (24.7 g) was dissolved in 200 ml of refluxing t-butanol. A mixture of isobutyraldehyde (18.2 ml) and diethyl succinate (41.55 ml) was added to the t-butanol solution over 30 min. The reaction mixture was stirred under reflux for 2 h, solvent was removed, and the residue was acidified with 2 N HCl. The product was extracted with ethyl ether (Et(2)O; 3 times 200 ml). The ether solution was washed with water, and the product was extracted with 10% Na(2)CO(3). The solution was acidified with concentrated HCl, and the product was isolated by extraction with Et(2)O. The ether layer was washed with water, dried over sodium sulfate, and evaporated. The remaining oil was dissolved in ethanol and hydrogenated in the presence of 10% palladium on charcoal. The resultant mixture was filtered through Celite, and the solvent was evaporated to yield 38.04 g of 2-carboethoxycarbonyl-4-methyl pentanoic acid as a mixture of isomers in the form of an oil. The isolated monoethyl ester was further purified on silica gel (Kieselgel 60, 320-400 mesh) by flash chromatography using ethyl acetate/hexane/acetic acid (1:10:0.5, v/v/v). NMR: (300 MHz, CDCl(3)): (ppm) 0.90 (6H, q, (CH(3))(2)CH); 1.20-1.38 (4H, m, CH(3) ester +(CH(3))(2)CH); 1.56 (2 H, m, CH(2)-CH); 2.45 and 2.72 (2H, dd and q, HOOC-CH(2)); 2.84 (1H, m, CH(2)-CH-CO); 4.14 (2H, q, CH(2) ester).

Synthesis of 2-Carbo-t-butoxycarbonyl-4-methylpentanoic Acid

To a chilled (-70 °C) solution of 3-(ethoxycarbonyl)-3-(2-methylpropyl)propanoic acid (23 g) in 200 ml of methylene chloride was added isobutylene (200 ml) and concentrated sulfuric acid (4 ml). The reaction mixture was stirred in a glass medium pressure reaction vessel for 4 days at room temperature and then cooled to -70 °C and mixed with 80 ml of saturated NaHCO(3). Standard workup yielded the diester (29.48 g) as an oil. The diester was hydrolyzed with 2 N NaOH (40.5 ml) in 200 ml 50% aqueous ethanol for 12 h at room temperature. The ethanol was evaporated, and the remaining oil was diluted with water and extracted with ethyl ether. Following acidification and ether extraction, the obtained monoester (15.8 g) was purified on silica gel by flash chromatography using ethyl acetate/hexane/acetic acid (1:9:0.1, v/v/v). NMR: (300 MHz, CDCl(3)): (ppm) 0.90 (6H, q, (CH(3))(2)-CH); 1.27 (1H, m, (CH(3))(2)CH); 1.41 (9H, s, (CH(3))(3)-C); 1.60 (2H, m, (CH(3))(2)CHCH(2))); 2.34 and 2.55 (1H and 1H, dd and q, CO-CH(2)-CH); 2.82 (1H, m, CH-COOH).

Preparation of HONHCOCH(2)CH(CH(2)CH(CH(3))(2))CO-Nal-Ala-NH(2). 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(3) (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(2)SO - d(6)); (ppm) 0.64 and 0.73 (6H, 2xd, (CH(3))(2)CH); 0.90 (3H, m, (CH(3))(2)-CH-CH(2); 1.22 (3H, d, CH(3)-CH); 2.64 and 3.00 (1H and 1H, m, CH(2)-CH(7)); 1.84-1.96 (3H, m, CO-CH(2)-CH); 4.20 (1H, m, CH-CH(2)CH(3)); 6.984 and 7.21 (1H and 1H, s, CO-NH(2)); 7.40-7.90 (8H, m, CH(7) and CH(2)-CH-CO-NH(2)); 8.18 (1H, d, CO-NH-CH-CH(3)); 8.66 (1H, s, NH-OH). FABMS: calc. (M+H) 457.23, found 457. HPLC (Isomer I): Rt = 13.5 min, Hibar C(18) (4.7 times 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(2)SO - d(6)); (ppm) 0.24 (3H, d, (CH(3))(2)CH); 0.38 (4H, m, (CH(3))(2)CH and (CH(3))(2)CH); 0.70 and 1.08 (1H and 1H, m, (CH(3))(2)-CH-CH(2)); 1.34 (3H, d, CH(3)-CH); 1.92 and 2.02 (1H and 1H, s, CO-CH(2)-CH); 2.86 and 3.34 (1H and 1H, m, CH(2)-CH(7)); 2.47 (1H, m, CH(2)-CH-CO); 4.23 (1H, m, CH-CH(7)); 4.55 (1H, m, CHCH(3)); 7.02 and 7.15 (1H and 1H, s, CO-NH(2)); 7.44-7.78 (7H, m, CH(7)); 8.00 (1H, d, CH(2)CO-NH-); 8.42 (1H, d, CO-NH-CH-CH(3)); 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(18) (4.7 times 150 mm), gradient 30-60% B in 30 min, A: 0.05% TFA; B: 0.05% TFA in AcCN, 1 ml/min.


RESULTS

An L-Selectin-Alkaline Phosphatase Reporter Construct

To facilitate the screening of protease inhibitors, we constructed an alkaline phosphatase reporter system to measure proteolysis at the L-selectin cleavage site. Our previous results indicated that the membrane-proximal cleavage domain was necessary to confer proteolysis(9) . We fused the 5` sequences encoding the signal sequence and ectodomain of the human alkaline phosphatase gene in-frame with 3` sequences of the L-selectin cDNA encoding the membrane-proximal cleavage domain, the transmembrane domain, and cytoplasmic domain (Fig. 1A). The L-AP chimera was expressed transiently in COS cells (data not shown) and stably in the L1.2 pre-B cell line (Fig. 1, B-D). Cell surface expression was verified by staining with an anti-human placental alkaline phosphatase mAb and analyzed by flow cytometry (Fig. 1B). Specific staining was detected on the L-AP-transfected cells but not on the mock-transfected parent cell line. Expression of the L-AP reporter was down-regulated upon activation with PMA (Fig. 1B) in a manner that paralleled down-regulation of L-selectin on PMA-stimulated L-selectin-transfected L1.2 cells. To further substantiate that the L-AP was expressed and cleaved as expected, the L-AP reporter was analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. L-AP-transfected L1.2 cells were metabolically labeled with [S]cysteine and [S]methionine. Cell lysates were immunoprecipitated with anti-alkaline phosphatase antibody or with JK564 antiserum directed against the cytoplasmic domain of L-selectin. Both antibodies immunoprecipitated the full-length membrane-bound chimera of 80 kDa (Fig. 1C, lanes 2, 3, 5, and 6). A soluble 74-kDa fragment of alkaline phosphatase was specifically immunoprecipitated with anti-alkaline phosphatase antibody from cell-free supernatants of L-AP-transfected L1.2 cells (Fig. 1C, lanes 8 and 9).

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).

Proteolysis of the L-AP Reporter Is Inhibited by Peptide Hydroxamic Acids

We tested a series of peptide hydroxamic acids, which were originally synthesized as inhibitors of zinc-dependent matrix metalloproteases. KD-IX-73-4 and KD-IX-73-5 are two diastereomers (Fig. 2A). KD-IX-73-4 is a potent collagenase and gelatinase inhibitor, whereas the diastereomer KD-IX-73-5 is much less active against matrix metalloproteases(20) . KD-IX-73-4 inhibited L-AP proteolysis in a dose-dependent manner (Fig. 2B) and reduced L-AP release to near background levels at 25-50 µg/ml. The IC of inhibition with KD-IX-73-4 was approximately 4.5 µM. In contrast, the diastereomer KD-IX-73-5 was at least 20-25-fold less potent than KD-IX-73-4. Alkaline phosphatase is a metal-dependent enzyme whose activity is inhibited by EDTA. Indeed 5 mM EDTA inhibits the L-AP reporter assay (Table 1). To determine if KD-IX-73-4 specifically inhibits the putative secretase or nonspecifically inhibits the activity of the alkaline phosphatase reporter, we tested the effects of EDTA and KD-IX-73-4 on exogenous soluble human placental alkaline phosphatase. EDTA completely inhibited alkaline phosphatase activity, as expected (Table 1). However KD-IX-73-4 had no significant effect on exogenous alkaline phosphatase activity, indicating that it did not nonspecifically inhibit activity of the reporter enzyme.


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.





The Hydroxamic Acid-based Peptide Inhibits Lymphocyte L-Selectin Shedding

We next tested whether the peptide hydroxamic acid compounds would inhibit down-regulation of wild type L-selectin from peripheral blood lymphocytes. Lymphocytes were pretreated with KD-IX-73-4 or the diastereomer KD-IX-73-5 for 10 min and then activated with 100 ng/ml PMA. KD-IX-73-4 inhibited L-selectin down-regulation in a dose-dependent manner in response to PMA stimulation, as judged by cell surface expression of L-selectin (Fig. 3). The IC of inhibition was approximately 3 µM. Pretreatment of lymphocytes with 50 µg/ml KD-IX-73-4 followed by washing the compound out prior to activation had minimal effects on subsequent down-regulation of L-selectin (Fig. 3). These results suggest that the protease is protected from the action of the compound until cell activation or that the compound readily dissociates from the protease. The less active diastereomer KD-IX-73-5 had partial activity at 50 µg/ml and no appreciable activity at low concentrations. In contrast, 5 mM EDTA had no effect on L-selectin shedding (Table 1) as described previously(11, 19) .


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.



The L-Selectin Protease Activity Acts in cis with Membrane-bound L-Selectin

We next examined whether the protease activity is likely to be a soluble secreted enzyme or a membrane-bound enzyme. We incubated neutrophils and lymphocytes together at a ratio of 7:1 and stimulated the cells with 100 ng/ml PMA or 10M fMLP. Both lymphocytes and neutrophils responded to PMA by down-regulating L-selectin expression (Fig. 5). However fMLP, which stimulates neutrophils but not lymphocytes, caused L-selectin down-regulation selectively on the neutrophil population. These results indicate that the putative secretase acts in cis with membrane-bound L-selectin and suggest that it is not a freely soluble activity.


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 times 10^7 cell/ml. The cell mix was left unstimulated or was stimulated with 50 ng/ml PMA or 10M 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.




DISCUSSION

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-alpha 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-alpha processing is similar to what we found for L-selectin proteolysis. A similar compound, TNF-alpha protease inhibitor, has also been found to inhibit release of the TNF receptor(24) . Notably TNF-alpha processing, in contrast to L-selectin proteolysis, is inhibited by EDTA(21) . Moreover TNF-alpha is a type II protein, whereas L-selectin is a type I protein. L-Selectin and TNF-alpha are among an emerging class of diverse proteins, such as transforming growth factor-alpha (25, 26, 27) , beta-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. (^2)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Boehringer Ingelheim Pharmaceuticals, Dept. of Immunological Diseases, R6-5, 900 Ridgebury Rd., Box 368, Ridgefield, CT 06877. Tel.: 203-798-4651; Fax: 203-791-6196.

(^1)
The abbreviations used are: L-AP, L-selectin-alkaline phosphatase; mAb, monoclonal antibody; PMA, phorbol myristate acetate; PHA, phytohemagglutinin; TNF, tumor necrosis factor; fMLP, formylmethionylleucylphenylalanine; HPLC, high pressure liquid chromatography.

(^2)
B. Walcheck, J. Kahn, C. Feehan, R. Betageri, K. Darlak, A. F. Spatola, and T. K. Kishimoto, submitted for publication.


ACKNOWLEDGEMENTS

We are grateful to the Boehringer Ingelheim Pharmaceuticals, Inc. biotechnology group for scale-up and purification of mAb, Carol Stearns for analysis with the FACScan, Liz Mainolfi for assistance with the luminometer, Patty Reilly for oligonucleotide synthesis, Anthony Shrutkowski and Dr. John Miglietta for operation of the DNA sequencer, and Grace Migaki, Kathy Last-Barney, and Dr. Robert Rothlein for critical comments, discussions, and support.


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