activation of paneth cell -defensins in mouse small intestine

Activation of Paneth Cell -Defensins in Mouse Small Intestine

Tokiyoshi Ayabe‡*

1, Donald P. Satchell*

1, Patrizia Pesendorfer

3, Hiroki Tanabe

1, Carole L. Wil-

son4, Susan J. Hagen

5, and Andre J. Ouellette

1,2¶

Departments of 1Pathology and

2Microbiology & Molecular Genetics, University of California

College of Medicine, Irvine, CA 92697-4800, 3Department of Pediatric Surgery, Karl-Franzens-

Univerity Graz, Graz, Austria, 4Division of Allergy and Pulmonary Medicine, Department of

Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, and 5Department

of Surgery, Beth Israel Deaconess Medical Center, 300 Brookline Avenue, Boston, MA 02115

*Equal contributors to the study.

‡Current address:

Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan

Running title: Intracellular processing of mouse procryptdins

¶Corresponding author: Andre J. Ouellette, Ph.D., Department of Pathology,

College of Medicine, University of California, Irvine, CA 92697-4800.

Tel: (949)824-4647, Fax: (949)824-1098, E-mail: [email protected]

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ABSTRACT

Paneth cells in small intestinal crypts secrete microbicidal -defensins, termed cryptdins,

as components of enteric innate immunity. The bactericidal activity of cryptdins requires prote-

olytic activation of precursors by the matrix metalloproteinase matrilysin (MMP-7a, Wilson et

al., Science: 286, 113-117, 1999). Here, we report on the intracellular processing of cryptdin

proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural

procryptdins identified conserved cleavage sites in the proregion between Ser43 and Val44 as

well as at the cryptdin peptide N-terminus between Ser58 and Leu59. Immune staining co-

localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing

evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Pa-

neth cells as shown by western blot analyses of intestinal crypt proteins and proteins from gran-

ule-enriched subcellular fractions. The addition of soluble prosegment to in vitro antimicrobial

peptide assays inhibited the bactericidal activities of cryptdins 3 and 4 in trans, suggesting pos-

sible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins are

normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine

grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not re-

quire direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is suffi-

cient to process and activate -defensin precursors. MMP-7-dependent procryptdin activation in

vivo provides mouse Paneth cells with functional peptides for apical secretion into the small in-

testinal lumen.


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INTRODUCTION

The release of endogenous antimicrobial peptides by mammalian epithelial cells contrib-

utes to innate mucosal immunity (1,2). The crypts of Lieberkühn in the small intestine of most

mammals contain Paneth cells that secrete -defensins (cryptdins), lysozyme, sPLA2, xanthine

oxidase, CD95 ligand, CD15, and TNF- as components of apically-oriented secretory granules

(3-10). Although certain Paneth cell -defensins have been detected in mouse skin and testis

(11,12) and in human oropharyngeal and urogenital mucosa (13,14), in the small intestine, -

defensins are specific to Paneth cells (9). Exposure of Paneth cells to cholinergic agonists or

bacterial stimuli elicits granule discharge into the crypt lumen (15), and carbamyl choline medi-

ates secretion via increased cytosolic Ca2+

(16). Regardless of how mouse Paneth cell secretion

is stimulated, cryptdins constitute ~70% of the released bactericidal activity, and the concentra-

tion of cryptdins is estimated to be 25 mM at the point of secretion in the crypt lumen (15).

-Defensins are processed from inactive proforms by specific proteolytic cleavage steps.

Both neutrophil and Paneth cell -defensins derive from ~10 kDa prepropeptides that contain

canonical signal sequences, acidic proregions, and a ~3.5 kDa mature -defensin peptide in the

C-terminal portion of the precursor. For example, maturation of myeloid pro -defensins ap-

pears to involve two primary cleavage steps, and most -defensins in mature phagocytic leuko-

cytes are completely processed (17-20). In a heterologously-expressed human neutrophil pro- -

defensin, deletions in the prosegment adjacent to the proregion-defensin junction impaired post-

translational processing in 32DCL3 cells (19).

In mouse Paneth cells, the matrix metalloproteinase matrilysin (MMP-7) mediates the

processing and activation of -defensins from 8.4 kDa proforms (21). MMP-7 gene disruption

ablates procryptdin processing, resulting in accumulation of cryptdin precursors and the absence


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of activated, mature cryptdin peptides in the small intestine (21). Lacking functional cryptdin

peptides, MMP-7-null mice have a defect in clearance of intestinal infections, and they succumb

more rapidly and to lower doses of virulent Salmonella typhimurium compared to control mice

(21). Thus, the cryptdin deficiency resulting from defective procryptdin activation is associated

with a measurable deficit in mucosal immunity and increased risk of systemic disease.

In this study, cryptdin biosynthesis was investigated by characterizing details of intracel-

lular procryptdin processing in mouse Paneth cells. The products of in vitro cleavage of

procryptdin-1 and natural procryptdins by MMP-7, the localization of the cryptdin proregion in

the exocytotic pathway, and the extent of procryptdin activation in Paneth cells of adult mice

have now been characterized. Our results show that extensive intracellular procryptdin activa-

tion occurs in mouse Paneth cells and that exposure to bacterial antigens does not induce

procryptdin processing in mice.

EXPERIMENTAL PROCEDURES

Animals and Tissue Preparation -- All procedures on mice were performed in compli-

ance with the policies of the Institutional Animal Care and Use Committee of the University of

California, Irvine. Outbred Swiss mice [(Crl:CD-1)(ICR)BR], 45 day old males, 6 week-old

adult male BALB/cJ and C57/Bl6 mice, 6 week-old pregnant female BALB/cJ mice, and 6

week-old adult male, germ-free Swiss mice were purchased from Charles River Breeding Labo-

ratories, Inc. (North Wilmington, MA). Matrilysin (MMP-7) null mice were 6 to 8 week-old

males backcrossed for 10 generations onto the C57/BL6 background. Mice were housed under

12 h cycles of light and dark and had free access to standard rat chow and water.


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For preparation of fetal small intestinal implants, pregnant BALB/cJ mice were eutha-

nized at 15-17 days gestation by injection with 600 g/g body weight of Avertin (500 mg tri-

bromoethanol, 250 mg 2-methyl-2-butanol in 39.5 ml water). Segments (1-2 cm) of proximal

small intestine from each fetus were implanted aseptically under dorsal subcutaneous skin flaps

of individual 6 week old isogenic BALB/cJ male mice (22,23). Approximately 90% of implants

grew and were harvested for isolation of RNA or protein, or they were fixed by immersion in

phosphate-buffered formalin. Fixed tissue was processed into paraffin blocks, sectioned and

stained with hematoxylin-eosin by the Department of Pathology Histology Lab, UCI Medical

Center.

Preparation of small intestinal crypts -- Crypts were prepared by EDTA treatment of

everted small intestinal segments as described (15,24,25,26). Briefly, segments adult mouse

small bowel were agitated in buffered 30 mM EDTA (pH 7.4), and eluted crypts were deposited

by centrifugation and resuspended in ice-cold Ca++

, Mg++

-free buffer. Enteric -defensins derive

exclusively from Paneth cells in crypts (15,27,28). Certain experiments were conducted with

Protease Inhibitor Cocktail Set III from CalBiochem (Costa Mesa, CA) present in all buffers and

solutions to test for the possibility of proteolysis during sample preparation. After protease in-

hibitors were shown to have no effect on procryptdin recovery or on the state of cryptdin activa-

tion (Fig 4C), experiments were conducted in the absence of inhibitors.

Extraction of crypt proteins -- Peptides were prepared by extraction using 30% acetic

acid (28,29). For analysis of peptides from crypt-enriched fractions, crypts were resuspended in

30% acetic acid, sonicated, and extracted overnight at 4oC. Extracts were centrifuged 15 min at

10,000 rpm in the Sorvall SA-600 rotor, supernatants were clarified by centrifugation for 2 h at


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28,000 rpm in the Beckman SW28.1 rotor, and high speed supernatants were diluted 10-fold and

lyophilized (21).

Preparation of Paneth cell secretory granules -- Subcellular fractions enriched for Pa-

neth cell secretory granules were prepared from duodenal and ileal crypts. Crypts deposited by

centrifugation at 700 rpm for 5 min in the Beckman GS-6R centrifuge were resuspended in ~10

ml ice-cold Ca++

, Mg++

-free phosphate-buffered saline (PBS), pH 7.5 (GIBCO, Gaithersburg,

MD), and placed under N2 at 750 psi for 15 min in a Model 1019HC nitrogen cavitation bomb

(Parr Instrument Company, Moline, IL). Cell lysates produced by equilibration to atmospheric

pressure were diluted two-fold with PBS containing 5 mM EDTA and centrifuged at 700 x g for

10 min at 4oC. Low-speed supernatants were reserved, and the deposited cell debris was washed

by resuspension in ice-cold Hanks-EDTA (GIBCO) and centrifugation at 700 x g for 10 min at

4oC. Granules in the combined supernatants were deposited by centrifugation at 27,000 x g for

40 min at 4oC in the Sorvall SA-600 rotor, and granules in the high-speed pellet were washed 2-3

times by resuspension and centrifugation in Hanks-EDTA under the same conditions. Granules

were stored frozen or dissolved immediately in 30% acetic acid and extracted as described

above.

Acid-Urea Polyacrylamide Gel Electrophoresis -- Lyophilized peptide samples were dis-

solved in 20 l of 5% acetic acid containing 3.0 M urea, and electrophoresed on 12.5% acid-urea

polyacrylamide gels (AU-PAGE) for 6 h at 150 V (29). Resolved proteins were visualized by

staining with Coomassie R-250 after fixation in formalin-containing acetic acid-methanol. -

Defensins were identified by their rapid co-migration with authentic mouse cryptdin peptides in

AU-PAGE (> 0.6 x Rf of methyl green dye) as described (30) and confirmed immunochemically

in western blots (15,31).


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Cryptdin-1 prosegment antiserum -- The cryptdin-1 prosegment corresponds to residues

19-58 in preprocryptdin-1 as deduced from cryptdin-1 cDNA (32,33, Fig 1A). The prosegment

(DPIQNTDEET KTEEQPGEDD QAVSVSFGDP EGTSLQEES) was synthesized by Quality

Controlled Biochemicals, Inc., (QCB, Hopkinton, MA). The composition and concentration of

synthetic prosegment was determined by amino acid analysis on a Waters Model 2690 Alliance

Analyzer, and its mass was verified by matrix-assisted laser desorption ionization mode time-of-

flight mass spectrometry (MALDI-TOF MS) on a Voyager-DE instrument (PE-Biosystems, Fos-

ter City, CA) in the UCI Biomedical Protein and Mass Spectrometry Resource Facility. QCB

produced polyclonal anti-cryptdin-1 prosegment antiserum in a sheep by administering 4 dorsal

subcutaneous injections of prosegment conjugated to bovine serum albumin in complete Freund's

adjuvant. Injections were repeated twice, and the antiserum titer was evaluated by ELISA by

QCB. The primary structures of prosegments in all mouse defensin family precursors are highly

conserved (Fig 1A), and the antibody is likely to cross-react with all mouse defensin family pre-

cursors. Rabbit antisera to the CRS1C-1 prosegment (34,35, Fig 1A) react with mouse Paneth

cells specifically (36).

Immunolocalization of cryptdins and prosegments -- Immunoperoxidase staining was

performed by the Department of Pathology Histology Lab at the UCI Medical Center. Paraffin

sections of formalin-fixed mouse small bowel were deparaffinized with xylenes, treated for 30

min with 0.3% H2O2 and washed with water and PBS. Slides were incubated 3 times for 5 min

each in a microwave oven with antigen unmasking solution (Vector Laboratories, Inc., Burlin-

game, CA) then cooled in unmasking solution (Vector) for 30 min at room temperature. After

rinsing with PBS, sections were blocked by incubation with normal goat serum for 30 min, with

Avidin D blocking solution for 15 min, rinsed briefly with PBS, and then incubated with biotin


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blocking solution (Vector) for 15 min. Slides were incubated with a 1:100 dilution of sheep

cryptdin-1 prosegment immune antiserum or with serum from the sheep prior to immunization.

After 30 min, slides were washed 3X with PBS, incubated 30 min with a 1:2000 (28) dilution of

biotinylated donkey anti-sheep IgG and washed as before. After 60 min incubation with

VECTASTAIN ABC peroxidase reagent (Vector), slides were washed, flooded with diamino-

benzidine, washed and counterstained before mounting.

For immunogold co-localization of mature peptide and cryptdin-1 prosegment, samples

of jejunum were fixed with 2% formaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer

(pH 7.4) and embedded in Unicryl (Ted Pella, Inc., Redding CA) at -20oC. Thin sections placed

on formvar and carbon-coated grids were stained with rabbit anti-cryptdin-1 antibody (28),

washed, and reacted with a 1:25 dilution of protein A labeled with 10 nm gold (Ted Pella) as de-

scribed (27). Next, sections were incubated with sheep anti-cryptdin-1 prosegment immune IgG

(1:200) overnight at 4oC. After washing, sections were incubated with donkey anti-sheep IgG

conjugated to 20 nm gold (Ted Pella). Sections were counterstained with uranyl acetate and lead

citrate, examined in a JEOL 100 CX electron microscope and photographed. Equivalent dilu-

tions of preimmune sera provided negative controls in all experiments.

Purification of mouse procryptdins – Recombinant procryptdin-1 was prepared as de-

scribed previously (21). Briefly, mouse procryptdin-1 cDNA cloned in pMalc2 (New England

Biolabs Inc. Beverly, MA) was expressed as an MBP-fusion protein in E. coli BL21(DE3)

CodonPlus cells (Stratagene, La Jolla, CA) that was purified by amylose resin affinity chroma-

tography. Procryptdin-1, released from the fusion protein by digestion with 1 g Factor Xa per

mg fusion protein at 30 C for 48 h (New England Biolabs, Inc., Beverly MA), was separated


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from MBP by C-4 RP-HPLC (Vydac 214TP1010 column, Vydac, Hesperia, CA) and purified to

homogeneity by analytical C-18 RP-HPLC on a Vydac 218TP54 column (21).

For purification of mouse enteric procryptdins, small intestinal protein extracts were pre-

pared from MMP-7 null mice by extraction with 30% acetic acid as described above. Protein

samples were applied to analytical C-18 RP-HPLC columns (Vydac 218TP54) in aqueous 0.1%

trifluoroacetic acid (TFA) and eluted at ~35 min using a 10 to 45% acetonitrile gradient devel-

oped over 55 min. Protein fractions containing apparent procryptdins were analyzed by acid

urea polyacrylamide gel electrophoresis (AU-PAGE) as described (21,29). Procryptdins A-C

were purified to homogeneity by C-18 reverse phase HPLC using a 120 min, 10-40% acetonitrile

gradient, from which cryptdin precursors eluted between 18-30 % acetonitrile (data not shown).

The identification of the purified proteins as cryptdin precursors was achieved by N-

terminal sequencing and MALDI-TOF MS. Peptide concentrations were determined using the

Bradford assay (Bio-Rad Laboratories, Hercules, CA), and the molecular masses of purified pu-

tative procryptdins were determined by MALDI-TOF MS followed by sequencing in the UCI

Biomedical Protein and Mass Spectrometry Resource Facility.

MMP-7 cleavage of mouse procryptdins in vitro -- Recombinant procryptdin-1 and natu-

ral procryptdins were digested with MMP-7, analyzed by AU-PAGE and SDS-PAGE, and mix-

tures of proteolytic digests from MMP-7 cleavage were analyzed by N-terminal sequencing.

Samples (1 g) of recombinant procryptdin-1 (21) and of natural procryptdins A, B, and C puri-

fied from MMP-7-null mice were incubated with equimolar quantities of activated recombinant

human MMP-7 catalytic domain (Chemicon International, Inc., Temecula, CA) in buffer

containing 10 mM N-[2-hydroxyethyl]piperizine-N'-[2-ethanesulfonic acid] (HEPES, pH 7.4),

150 mM NaCl, 5 mM CaCl2 for 24 h at 37oC. Reactions were analyzed in 15% polyacrylamide

Tris-Tricine SDS-PAGE gels (BioRad Laboratories, Hercules, CA). Curiously, proregions or


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Tricine SDS-PAGE gels (BioRad Laboratories, Hercules, CA). Curiously, proregions or frag-

ments of proregions are not seen by routine gel staining methods after digestion with MMP-7,

even though procryptdins and cryptdin peptides stain well (Fig 3A, D.P.S. & A.J.O., unpub-

lished). Samples (~ 200 ng) of complete digests were subjected to 8 cycles of N-terminal pep-

tide sequencing at The UCI Biomedical Protein and Mass Spectrometry Resource Facility.

Western blot analyses of Paneth cell -defensin precursors – Proteins extracted from

adult outbred Swiss mouse crypts were resolved by AU-PAGE, transferred to 0.2 m nitrocellu-

lose membranes, blocked, and incubated with sheep anti-cryptdin-1 prosegment immune IgG

diluted 1:2000 in TBST + 5% nonfat milk at room temperature with agitation (21). Washed

blots were incubated with peroxidase-conjugated donkey anti-sheep Ab diluted 1:5000 in TBST

for 30 min, washed and developed using SuperSignal chemiluminescent substrate (Pierce, Rock-

ford, IL) with a 10-15 min exposure (21). In western blots using rabbit anti-cryptdin-1 peptide

antiserum, goat anti-rabbit IgG was used as the secondary antibody at a 1:20,000 dilution (15).

Assays of Bactericidal Peptide Activity –To measure bactericidal activities, ~1 x 106 ex-

ponentially-growing Escherichia coli ML35 cells were incubated with 5 g/ml synthetic

cryptdin-3 or recombinant cryptdin-4 in 10 mM PIPES buffer (pH 7.4) with quantities of

prosegment, corresponding to preprocryptdin-1 residues 19 to 58 (Fig 1A). After 60 min at 37oC,

20 l of each incubation mixture was diluted 1:2000 with 10 mM PIPES, pH 7.4, and 50 l of

the diluted samples were plated on trypticase soy agar using a Spiral Biotech Autoplate 4000

(Spiral Biotech Inc., Bethesda, MD). Surviving bacteria were quantitated as colony forming

units (CFU/ml) on plates after incubation at 37 C for 12 h.

RESULTS


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Cryptdin-1 prosegment in mouse Paneth cells – A polyclonal sheep antibody raised

against full-length synthetic cryptdin-1 prosegment (Fig 1A) reacts specifically with

procryptdin-1 and with procryptdins in extracts of mouse small intestinal proteins (Fig 1). West-

ern blot analysis (Experimental Procedures) showed that the antibody was specific for

procryptdins in intestinal protein extracts, which co-migrated with recombinant procryptdin-1

(21, Fig 1B). Because mouse defensin family proregions have extensive sequence similarity (Fig

1A), these data are probably a measure of immunoreactivity with procryptdin-1 and with the

many defensin and defensin-related precursors expressed by mouse Paneth cells (33-35,37).

In small intestine, cryptdin transcripts and peptides previously have been found only in

Paneth cells (9,15,27,28,38-42), and consistent with those findings, immunoperoxidase detection

of the cryptdin-1 prosegment showed that it also is Paneth cell-specific (Fig 1C). This finding is

in agreement with immunolocalization of the related mouse CRS1C prosegment (Fig 1A) using a

rabbit polyclonal antibody to the CRS1C-1 proregion (36, A.J. Ouellette, unpublished). The re-

active cryptdin prosegment antigen appeared to be associated with secretory granules, prompting

immunolocalization studies at the electron microscopic level.

Cryptdin precursors in Paneth cell secretory granules -- -Defensin prosegments as well

as cryptdin peptides are constituents of mouse Paneth cell secretory granules. The subcellular

location of cryptdin prosegments within Paneth cells of mouse mid small bowel was determined

using appropriate gold-conjugated protein A or second antibodies. As shown in Figure 2A-C,

both the prosegment and the cryptdin peptide antibodies reacted strongly and specifically with

Paneth cell granules. Preimmune negative control sera had very low background staining (Fig

2C, inset). With both prosegment and cryptdin peptide antisera, the respective antigens first

were detected in the trans-Golgi of the Paneth cell exocytotic pathway (Fig 2B, inset). Cyto-


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plasmic staining was highly specific for the electron-dense region of secretory granules (Fig

2B,C). The electron-lucent haloes of Paneth cell granules, which contain high levels of GalNAc-

O-linked glycoconjugates (43), showed very little gold staining (Fig 2B,C). All granules were

immunoreactive, and staining was uniformly equivalent regardless of subcellular localization, as

shown by quantitation of gold particles over apical or supranuclear granules (Fig 2D). Despite

showing that the prosegment antibody reacted with Paneth cell granules, these findings did not

distinguish unprocessed procryptdins from soluble proregions generated by MMP-7 proteolysis

of cryptdin precursors. To resolve this question, the products of in vitro MMP-7 hydrolysis of

procryptdins in vitro and the status of procryptdin activation in Paneth cells in vivo were investi-

gated in detail.

Specificity of in vitro procryptdin cleavage by MMP-7 – Because Paneth cell -defensin

processing intermediates had not been characterized, mouse procryptdins were purified from

MMP-7 null mouse small intestine as substrates for analysis of the MMP-7 cleavage products.

MMP-7 null mice are an optimal source for cryptdin precursor purification, because cryptdin

gene expression occurs at wild-type levels, and procryptdins accumulate in MMP-7 deficient Pa-

neth cells (21). Also, studies of natural substrates avoid potential complications of analyzing

possibly misfolded recombinant cryptdin precursors.

Putative mouse procryptdins A, B, and C were purified to homogeneity by combined C-4

and C-18 RP-HPLC (Fig 3A, Experimental Procedures). Candidate molecules were deduced to

be cryptdin precursors based on elution times from C18 RP-HPLC and co-migration with re-

combinant procryptdin-1 in SDS- (Figs 1B, 3A) and AU-PAGE (not shown). MALDI-TOF MS

of putative procryptdins A-C provided atomic masses of 8543, 8478, and 8277 a.m.u., respec-

tively, values that did not correspond to previously deduced procryptdin sequences (32,33,38).


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Despite this apparent discrepancy, procryptdins A-C were shown to be -defensin precursors by

N-terminal sequencing, because they had N-termini identical to that of procryptdin-1, DPIQNTD

(Table), the consensus N-terminus of all known mouse procryptdins (32,33,38, Fig 1A). Analy-

sis of procryptdins A-C by SDS-PAGE following cleavage with MMP-7 in vitro produced only

one evident primary cleavage product of appropriate mobility for mature -defensin peptides

(Fig 3A).

The peptide bonds cleaved in procryptdins A-C by MMP-7 were determined by direct N-

terminal sequencing of MMP-7 digests of the precursors (Fig 3B). For each putative

procryptdin, only three N-termini were detected besides that of the activated MMP-7 enzyme

(Table, Fig 3B). The first N-terminal sequence was DPIQNTD..., the consensus procryptdin N-

terminus (Table). The second sequence was VSFGDPEG..., an internal cleavage site between

Ser43 and Val44 in the prosegment (Table, Fig 3B). The V-S-*-V-S-F-G sequence flanking the

cleavage site (“*”, Fig 3B) within the prosegment is conserved in all mouse defensin family pre-

cursors (32,33,37, Fig 1A). The third sequence was LRDLV_Y_..., where underscore characters

represent deduced cysteines, and that N-terminal sequence results from proteolysis between

Ser58 and Leu59 in all related procryptdins (21, Fig 3B). LRDLV is the consensus N-terminus

for all cryptdin peptides except cryptdins 4 and 5 (33). The masses determined for procryptdins

A-C were not in concordance with known mouse procryptdins, perhaps because the MMP-7

knockout is on the C57/Bl6 genetic background. Previous clones of procryptdin cDNAs and

genes were from inbred C3H/HeJ and 129/SvJ mouse strains or from outbred Swiss mice, and

these strains may have unreported proregion or cryptdin peptide amino acid substitutions that

differ from those in C57/Bl6 mice. Collectively, these results both confirmed procryptdins A-C


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as cryptdin precursors and defined an MMP-7 catalyzed processing site within the proregions of

these precursors.

Activated -defensins in mouse Paneth cell secretory granules – The colocalization of

prosegments and cryptdins in secretory granules (Figs 1C,2) prompted an evaluation of the proc-

essing status of cryptdin precursors in Paneth cell granules. The distribution of cryptdins and

procryptdins in Paneth cell secretory granules was determined by AU-PAGE western blotting

(15,31). In AU-PAGE, activated -defensins are the most rapidly migrating intestinal peptides

(Experimental Procedures), and they are lacking in MMP-7 null mice (21, Fig 4A). Previously,

only low levels of procryptdins were detected in secretions elicited from Paneth cells by carba-

myl choline exposure (15). Partially purified Paneth cell secretory granules contained abundant

activated cryptdins at levels equivalent to those in intact crypts (Fig 4B, lane 2). Inclusion of a

complex of potent proteinase inhibitors in all solutions and buffers during crypt isolation, gran-

ule sedimentation, and protein extraction (Experimental Procedures) had no effect on the appar-

ent levels of activated cryptdins (Fig 4C), a fact taken as evidence that procryptdin processing

was not caused by experimental manipulation.

The relative distribution of cryptdins to procryptdins was evaluated by western blot

analysis of Paneth cell granule proteins from wild-type and MMP-7 null mice using a cryptdin-1

peptide antibody (15,28). As predicted from previous analyses of whole mouse small bowel pro-

teins (21), Paneth cell granules from MMP-7 null mice lacked rapidly-migrating, activated

cryptdins but contained high levels of procryptdins (Fig 4D, lane 2). In contrast, granule pro-

teins from C57/Bl6 wild-type mice gave strong immunoreactivity at the position of cryptdin mo-

bility, where the signal strength was approximately twice that of the procryptdin region (Fig 4D).

From these considerations and because cryptdins give weaker immune staining in westerns than


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equimolar quantities of procryptdins (M.E. Selsted, personal communication), we estimate that

60-70% of the procryptdins in Paneth cells are processed to functional peptide before secretion

(see Discussion). Because extensive procryptdin activation is intracellular (Fig 4), prosegments

in granules (Fig 2) are subject to secretion, suggesting that proregions might inhibit the bacteri-

cidal activities of cryptdin peptides.

Soluble prosegment neutralizes cryptdin bactericidal activity in vitro – The cryptdin-1

prosegment lacks antimicrobial activity, but it inhibited the bactericidal activity of mature

cryptdin peptides in trans. The ability of soluble cryptdin-1 propeptide, corresponding to resi-

dues 19 to 58 in the cryptdin-1 precursor (Fig 1A), to inhibit the activity of cryptdins 3 and 4 was

tested in bactericidal assays against E. coli ML35. In agreement with the inhibition of myeloid

-defensins by human neutrophil proregions (20), prosegment to cryptdin molar ratios of 0.5 to 1

or greater inhibited both peptides to approximately the same extent (Fig 5). Perhaps as other au-

thors have suggested (17), the acidic proregions (pI ~ 3.4) may neutralize the activity of the cati-

onic defensins by charge neutralization. These in vitro inhibitory activities of secreted proseg-

ments in trans are consistent with a possible cytoprotective role.

Procryptdin activation in germ-free mice – Because MMP-7 is required for procryptdin

activation (21, Fig 4A), we evaluated the extent of cryptdin activation in Paneth cells of germ-

free mice to test whether their basal MMP-7 levels are adequate for cryptdin processing. Germ-

free mice contain less Paneth cell MMP-7 than conventional mice, and monocolonization of

germ-free mice with Bacteroides thetaiotaomicron induces expression of Paneth cell MMP-7 to

levels found in mice harboring conventional microflora (36). Whether raised conventionally or

germ-free, mouse intestinal extracts contained comparable levels of activated cryptdins (Fig 6),

and thus sufficient MMP-7 exists under germ-free conditions to activate cryptdins normally. Al-


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though the mice were germ-free and consumed sterile chow, exposure to dietary bacterial anti-

gens may have been responsible for inducing elevated MMP-7 levels, even in the germ-free

state. For that reason, the level of cryptdin activation was examined in sterile implants of fetal

mouse small intestine grown subcutaneously (22,23).

Activated cryptdins in implants of fetal small intestine -- To test whether procryptdin ac-

tivation requires exposure to bacterial antigens, the state of cryptdin processing was investigated

during Paneth cell ontogeny in BALB/cJ isogenic implants. In mice, the ontogeny of the small

intestinal epithelium occurs during the first three weeks postpartum (45,46). Interestingly, sub-

cutaneous growth of fetal intestinal implants provides conditions that favor epithelial cell differ-

entiation in structures that develop to resemble the morphology of normal adult small intestine

(22,23,47). In our experiments, ~ 90% of implants grew, and Paneth cells were evident at the

base of crypts by approximately 12 days after transplantation (PT 12) as judged by hematoxylin-

eosin staining. Paneth cell granules increased in number and size between PT 12 and PT 19 (Fig

7A). RT-PCR amplification assays for Paneth cell specific mRNAs in PT 7 to PT 28 implant

RNAs detected lysozyme, MMP-7, and cryptdin-1, 4, and 5 mRNAs at all time points (Fig 7B).

AU-PAGE analysis of proteins extracted from implants removed on PT 7 to PT 19 showed that

activated cryptdins were evident from PT 12 onward, resembling adult levels by PT 19. Protein

extracts from PT 7 implants and MMP-7-null mice lacked activated cryptdins (Fig 7). Because

Paneth cells in implants PT 12 or older contain processed cryptdins, luminal exposure to bacte-

rial antigens cannot be required to initiate procryptdin processing. Furthermore, Paneth cells that

are naive to luminal bacterial antigen exposure contain MMP-7 in adequate quantities to provide

functional cryptdins for secretion.


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DISCUSSION

In mouse small intestine, a substantial fraction of procryptdin activation occurs in Paneth

cells and prior to secretion. This conclusion is supported by evidence from electrophoretic and

western blot analyses of proteins extracted from subcellular fractions enriched in secretory gran-

ules, where 60-70% of procryptdins exist already activated by MMP-7-dependent proteolytic

cleavage (Fig 3). This value for the fraction of processed precursors represents an overall aver-

age for all cryptdin precursors, with the exception of cryptdins 4 and 5, which do not react with

the cryptdin-1 antibody. Interpretation of these data is complicated, though, by the dynamics of

crypt cell biology and by the ongoing processes of Paneth cell differentiation and granule bio-

genesis in the regulated secretory pathway. For example, our findings do not distinguish be-

tween the procryptdin activation state in mature granules poised for vesicular fusion at the Pa-

neth cell apical membrane from that in nascent granules that are forming in the trans-Golgi. In

addition, we cannot discount the possibility of post-secretory activation of the procryptdin mole-

cules that are secreted. Also, Paneth cells differentiate in crypts over ~ 8 days as they emerge

from the stem cell zone and descend to the base of the crypt (25). The extent of procryptdin

processing in granules of maturing Paneth cells may differ relative to that in fully differentiated

cells at the crypt base.

Questions remain regarding the biology of cryptdin prosegments. For example, the inhi-

bition of cryptdin bactericidal activity by addition of complete prosegment in trans (Fig 5) is

consistent with comparable dose-dependent inhibition of HNP-1 activity by recombinant HNP-1

prosegment [proHNP-120-64] (20) but paradoxical in view of the bactericidal activity of Paneth

cell secretions (15). Perhaps, as suggested for myeloid -defensin prosegments (20), cryptdin

propeptides may interact with additional chaperones to neutralize the potential membrane disrup-


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tive activities of mouse -defensins as they traverse the Paneth cell Golgi stack during granu-

logenesis. Because secretory granules containing activated cryptdins also react with prosegment

antibodies (36, Figs 1,2), the processed proregions or proregion fragments (Fig 3B) are likely to

be released along with activated cryptdins as Paneth cells degranulate. The high bactericidal

peptide activity in Paneth cell secretions (15), however, suggests that proregion inhibitory activ-

ity may be neutralized before or during secretion. Possibly, MMP-7 catalyzed proteolysis of

proregions between Ser43-Val44, Ser53-Leu54 (44), and Ser58-Leu59 during precursor activa-

tion may eliminate the inhibitory capabilities of the complete 39 amino acid proregion tested in

our studies. Also, MMP-7 cleaved procryptdins A-C reproducibly, but additional cleavage steps

may exist as suggested by the isolation of apparent procryptdin processing intermediates with

LQEESLRDLV N-termini from mouse small intestine (44). Those intermediates may be MMP-

7 cleavage products that our sequencing experiments did not detect, or they may be procryptdin

cleavage products of an MMP-7-dependent enteric protease(s) capable of cleaving the precursors

in vivo. Interestingly, preliminary studies of MMP-7 digests of recombinant procryptdin-4 have

not detected the proregion cleavage site between Ser42 and Ile43. Instead, an abundant LHEKS

N-terminal sequence was found, showing that MMP-7 cleaves procryptdin-4 between the Ala52

and Leu53 (Y. Shirafuji & A.J. Ouellette, unpublished), a site that corresponds to the intermedi-

ates purified by Putsep et al. (44). Thus, in vitro, MMP-7 appears to be capable of generating all

known cryptdin processing intermediates.

Paneth cells in germ-free mice have almost undetectable MMP-7 as previously deter-

mined immunohistochemically (36). Nevertheless, the base line level of MMP-7 suffices to in-

sure normal cryptdin activation (Figs 6, 7). Similarly, procryptdin processing in sterile intestinal

implants shows that enough MMP-7 exists (Fig 7B) to activate the pool of cryptdin precursors


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without microbial stimuli in the lumen (Fig 7C). Although RT-PCR amplification of implant

RNAs detected lysozyme, MMP-7, and cryptdin mRNAs in all implants (Fig 7B), cryptdin

RNAs were not detected by northern blot hybridization before PT 12 (data not shown). Similar

findings have been obtained in fetal and newborn mouse intestine, which also lacks Paneth cells

prior to crypt ontogeny and where cryptdins accumulate in apparent secretory cells of the matur-

ing epithelial monolayer (48).

Paneth cell differentiation inherent to small bowel development includes programmed

mechanisms for procryptdin activation and the secretion of functional -defensins without envi-

ronmental cues from the lumen. The evidence in support of this conclusion does not exclude re-

sponses of Paneth cells or their progenitors to proinflammatory mediators, including TNF- or

IFN- that may be released by neighboring epithelial cells or by stromal cells. In fact, Trichi-

nella spiralis infection of mouse small intestine stimulates an increase in Paneth cell numbers as

well as recruitment of intermediate cells to accumulate cryptdins in dense granules, and both

outcomes are mediated by T lymphocytes (49,50). Similarly, Paneth cells increase rapidly in

number when T cells are activated by CD3 ligation, and those events are partly dependent on

TNF- (51). Perhaps proinflammatory cytokines influence the inherent plasticity of the g

testinal epithelium by redirecting lineage determination programs in the short term and modulat-

ing Paneth cell numbers during inflammatory episodes. The responsiveness of MMP-7 biosyn-

thesis and activation to proinflammatory cytokines (52,53) is consistent with this possibi

Regardless of the mechanisms regulating MMP-7 expression in Paneth cells, MMP-7-dependent

procryptdin processing insures the secretion of active -defensins to facilitate innate mucosal

immunity.

astroin-

lity.


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ACKNOWLEDGMENTS

Supported by NIH grant DK10184 (D.P.S), DE14040 (C.L.W.), DK15681 (S.J.H.), and

DK44632 (A.J.O.). The thank Drs. Michael E. Selsted, Charles L. Bevins, Dipankar Ghosh, and

William C. Parks for useful discussions and Dana M. Frederick, Khoa Nguyen, Hao Truong, and

Hong Yang for excellent technical assistance. We thank Ms. Tracey Kingsley and Dr. Philip M.

Carpenter, Histology Lab, Department of Pathology, UCI Medical Center for performing histo-

chemical and immunoperoxidase experiments and Dr. Agnes Henschen, UCI Biomedical Protein

and Mass Spectrometry Resource Facility, for peptide sequencing and analysis.

aAbbreviation footnote: MMP-7, matrilysin; MALDI-TOF MS, matrix-assisted laser desorption

ionization mode time-of-flight mass spectrometry; RP-HPLC, reverse-phase high performance

liquid chromatography, RT-PCR, reverse transcriptase polymerase chain reaction; AU-PAGE,

acid urea polyacrylamide gel electrophoresis.


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LEGENDS TO FIGURES

Figure 1. Immunochemical analysis of mouse Paneth cell prosegments and -defensin precur-

sors. In A, alignment of prosegment sequences from mouse cryptdin and defensin-related pre-

cursors deduced from intestinal cDNAs (33,37) illustrates their extensive sequence similarity.

Numerals identifying residue positions are based on the deduced preprocryptdin-1 sequence with

position #1 at the initiating Met residue. Amino acid differences with the cryptdin-1 proregion

are noted in bolded characters. In B, antibody to cryptdin-1 prosegment is shown to react with

procryptdin-1 and procryptdins by SDS-PAGE western blot analysis of adult mouse small bowel

protein extracts (Experimental Procedures). PC-1 denotes a lane containing 1 µg of recombinant

procryptdin-1 (21), and the lane labeled “Gut” contained ~500 µg extracted peptides from adult

mouse small intestine (Experimental Procedures). In descending order, the bars at left represent

28, 18, 15.6, 7.6, and 3.55 kDa protein markers. The arrow at left denotes the position of immu-

noreactive procryptdins. In C, the Paneth cell -defensin prosegment was immunolocalized to

Paneth cell secretory granules (Experimental Procedures). Arrows indicate the presence of reac-

tive prosegment antigen in apparent secretory granules of Paneth cells.

Figure 2. Immunostaining of mouse Paneth cells with cryptdin and procryptdin antibodies. In

A, electron micrograph of an adult mouse small intestinal crypt. Paneth cells (P) reside at the

base of the crypt and are surrounded by undifferentiated crypt epithelial cells (C). Paneth cells

contain granules (G), an extensive system of rough endoplasmic reticulum (rER), and

prominent Golgi (Gp). L, Lumen of the crypt. Original magnification = 5,375X; Bar = 5 µm. In

B, the boxed supranuclear region in A is shown at higher magnification to visualize gold-labeled

structures after staining with rabbit anti-cryptdin and protein A gold (10 nm) and sheep anti-

prosegment and anti-sheep IgG gold (20 nm). Note that the Golgi (Gp) and granules (G) in the


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Paneth cell were labeled with both the 10 nm (short arrows) and 20 nm (long arrows) gold

probes, demonstrating that both peptides are present (inset). In contrast, the rough endoplasmic

reticulum (rER) is not labeled. Gold labeling of the Paneth cell Golgi (Gp) is much more exten-

sive than that of the Golgi (Gc) from adjacent undifferentiated crypt epithelial cells (C). Original

magnification = 22,306X. Bar = 1 µm. In C, a high magnification electron micrograph shows

co-localization of prosegment and cryptdin peptides in Paneth cell granules. This section was

labeled with rabbit anti-cryptdin-1/protein A gold (10 nm) and sheep anti-prosegment/anti-sheep

IgG gold (20 nm), and both 10 nm (short arrows) and 20 nm (long arrows) gold particles are pre-

sent in the electron dense (Gd), but not electron lucent (Gl), zones of Paneth cell granules. Inset

shows the relative lack of background staining in Paneth cell granules that were incubated with

preimmune sera prior to incubation with 10 and 20 nM gold-conjugates. Original magnification

= 53,750X. Bar = 0.5 µm. In D, sections labeled with rabbit anti-cryptdin-1/protein A gold (10

nm) were evaluated by counting gold particles in 38 apical and 40 supranuclear granules. The

labeling density of apical and supranuclear granules was identical. Data are presented as means

± S.E.M.

Figure 3. Recognition and cleavage of mouse procryptdins by MMP-7. In A, samples (1 µg) of

procryptdins A, B, and C, purified from MMP-7-null mice, were incubated overnight with (+) or

without (-) 2 µg MMP-7, and samples of digests were resolved by SDS-PAGE and stained with

Gel Code Blue (Pierce). Electrophoretic mobilities of individual components are noted at left in

descending order as follows: MMP-7, matrilysin; PC, purified procryptdins; Crp, MMP-7-

activated cryptdin peptides. Dashes at right denote, in descending order, the position of 28, 18,

15.6, and 7.6 kDa molecular weight markers. In B, the consensus cleavage sites disclosed by

protein sequencing of MMP-7 digests of procryptdins A-C (panel A) are noted by asterisks (*)


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that interrupt the procryptdin-1 sequence, and the pound character (#) shows the N-terminus of

procryptdin intermediates purified from mouse small bowel by Putsep et al. (44) that were not

evident in these in vitro analyses. Numerals above the primary structure refer to residue posi-

tions, with the initiating Met residue in preprocryptdin-1 as residue #1.

Figure 4. Intracellular processing of mouse Paneth cell -defensin precursors. In A: samples

(250 µg) of protein extracts from adult mouse small intestine were resolved by AU-PAGE and

gels were stained with Coomassie Blue (Experimental Procedures). Lanes 1 & 3, extracts from

MMP-7-null mice lack activated defensins (boxed region); lanes 2 & 4, extracts from wild-type,

C57/BL6 mice. In B, activated cryptdins are shown in proteins extracted from combined duode-

nal and ileal Paneth cell granules (Experimental Procedures) after resolution in AU-PAGE, and

stained with Coomassie Blue R250. Lanes: 1, extract from intact crypts; 2, granule extract; C1,

1 µg cryptdin-1; C3, 1 µg cryptdin-3; and C4, 1 µg cryptdin-4. In C, proteins extracted from s

retory granules prepared from adult mouse crypts in the absence (lane 1) or in the presence (lan

2) of Protease Inhibitor Cocktail III (CalBiochem, Experimental Procedures) were subjec

AU-PAGE. Equivalent quantities of protein were electrophoresed and the gel was stained with

Coomassie Blue. Lanes marked C1, C3, C4, contain 1 µg of cryptdin-1, 3, and 4, respectively.

In D, proteins from Paneth cell granules purified from wild-type (lane 1) or MMP-7 null (lane 2)

adult mouse small intestine were run in AU-PAGE, western blotted and probed with anti-

cryptdin-1 antibody. Lanes C1 and C3 contain 1 µg cryptdin-1 and cryptdin-3, respective

all panels, boxed regions denote the positions at which cryptdin peptides migrate in the AU

system. The arrow at right indicates procryptdins.

ec-

e

ted to

ly. In

gel

Figure 5. Cryptdin-1 prosegment neutralizes cryptdin bactericidal activities in trans. Synthetic

prosegment, corresponding to residues 19-58 in preprocryptdin-1 (Fig 1A), was combined with 5


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µg of cryptdin-3 (A) or cryptdin-4 (B) in the molar ratios shown, incubated with ~1 X 106 E. coli

ML35 cells for 60 min at 37oC, and surviving bacteria were determined by colony counts after

overnight growth on semi-solid media (Experimental Procedures). Bars labeled “C” denote bac-

terial survival in the absence of cryptdin peptides, and bars labeled Crp3 (in A) or Crp4 (in B)

show viability after exposure to 5 µg of cryptdin-3 or cryptdin-4, respectively, in the absence of

prosegment.

Figure 6. Activated cryptdins in germ-free mice. Proteins extracted from small intestines of

adult germ-free mice (lanes 1 & 2), mice conventionalized for 1d (lane 3) and 7 d (lanes 4 & 5),

and a conventionally-reared mouse (lane 6) were analyzed in an AU-PAGE gel and stained with

Coomassie Blue. The boxed region denotes the position of cryptdin peptides. Lanes C1, C3,

and C4 contain 1 µg of cryptdin-1, 3, and 4, respectively.

Figure 7. Activated cryptdins in fetal mouse intestinal implants grown subcutaneously. In A,

implanted tissue removed 5 to 28 days after implantation (PT 5 to PT 28) was fixed in buffered

formalin, processed, and stained with hematoxylin & eosin (Experimental Procedures). Arrows

indicate granule-containing Paneth cells in crypts of developed implants. In B, RNA from PT 7

to PT 28 implants was amplified by RT-PCR using primers specific for lysozyme, MMP-7,

cryptdin-1, cryptdin-4, and cryptdin-5 as reported previously (48,54). As in neonatal small

bowel (48), the Paneth cell marker mRNAs are present in the implanted tissues prior to the ap-

pearance of recognizable Paneth cells. Lanes marked “Ad” contain products amplified from to-

tal RNA from adult mouse small bowel, and lanes marked “W” contain equivalent samples of

amplification reactions in which water was substituted for template RNA. In C, samples (700

µg) of implant protein extracts were analyzed by AU-PAGE as in Fig 6. Lanes contain proteins

from implants taken 7 to 19 d after implantation (PT7 to PT19, respectively), intestinal protein


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extracts from MMP-7 null (-/-) and control wild-type (+/+) mice, respectively, and 1 µg

cryptdin-1 (C1) and cryptdin-3 (C3) as noted. The boxed region of the gel shows the position of

activated cryptdin peptides.


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19 30 40 50 58

Cryptdin-1 DPIQNTDEETKTEEQPGEDDQAVSVSFGDPEGTSLQEES

Cryptdin-2 DPIQNTDEETKTEKQPGEEDQAVSVSFGDPEGSSLQEES

Cryptdin-3 DPIQNTDEETKTEEQPGEDDQAVSVSFGDPEGSSLQEES

Cryptdin-4 DPIQNTDEETKTEEQPGEEDQAVSISFGGQEGSALHEKS

Cryptdin-5 DPIHKTDEETNTEEQPGEEDQAVSISFGGQEGSALHEE-

Cryptdin-6 DPIQNTDEETKTEEQPGEEDQAVSVSFGDPEGTSLQEES


Cryptdin-9 DPIQNTDEETKTEEQPGEEDQAVSVSFGDPEGSSLQEES






Cryptdin-14 DPIQNTDEETKTEEQPGEEDQAVSVSFGDPEGSSLQEES



CRS1C-1 DPIQNTDEETKTEEQPEEEDQAVSVSFGGTEGSALQDVA

CRS4C-1 DSIQNTDEETKTEEQPGEKDQAVSVSFGDPQGSALQDAA

Figure 1

A


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Figure 2

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Procryptdin-1

<------ Prosegment -------> <-- α-defensin peptide -->

19 43 44 53 54 58 59 92

DPIQNTDEETKTEEQPGEDDQAVS*VSFGDPEGTS#LQEES*LRDLVCYCRSRGCKGRERMNGTCRKGHLLYLCCR

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B

Figure 3

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Procryptdin: A B C

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1 2 3 4

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Figure 5

C Crp3 1:4 1:2 1:1 2:1

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Figure 6

1 2 3 4 5 6 C1C3C4


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AFigure 7

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PT 19

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B

Lysozyme

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Cryptdin-1

Cryptdin-4

Cryptdin-5

7 9 12 16 20 22 28 Ad W

12 12 15 20 W 22 22 28 Ad

7 9 12 16 20 22 28 Ad W

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Table1

MMP-7 Cleavage Sites in Mouse Cryptdin Precursors Protein Mass Amino Terminal Sequences N-terminus (A.M.U.) Undigested MMP-7 Digests Procryptdin-1 N.D. DPIQNTD DPIQNTD Asp19 VSFGDPEG Val44 LRDLV_Y_ Leu59 Procryptdin A 8543 DPIQNTD DPIQNTD Asp19 VSFGDPEG Val44 LRDLV_Y_ Leu59 Procryptdin B 8478 DPIQNTD DPIQNTD Asp19 VSFGDPEG Val44 LRDLV_Y_ Leu59 Procryptdin C 8277 DPIQNTD DPIQNTD Asp19 VSFGDPEG Val44 LRDLV_Y_ Leu59 1The molecular masses of putative natural cryptdin precursors were determined by

MALDI-TOF mass spectrometry, and N-terminal sequences were determined by Edman deg-

radation of samples before and after digestion with MMP-7 (Experimental Proce-

dures). N.D. = not determined. N-terminal residue assignments are based on the

primary structure of preprocryptdin-1 (Fig 4B) with residue #1 at the initiating

Met.

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Wilson, Susan J. Hagen and Andre J. OuelletteTokiyoshi Ayabe, Donald P. Satchell, Patrizia Pesendorfer, Hiroki Tanabe, Carole L.

Activation of Paneth cell alpha-defensins in mouse small intestine

published online December 3, 2001J. Biol. Chem.

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activation of paneth cell -defensins in mouse small intestine

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