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Published as doi: 10.1096/fj.07-103440.
 (The FASEB Journal. 2008;22:2427-2434.)
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Identification of heparin/heparan sulfate interacting protein as a major broad-spectrum antimicrobial protein in lung and small intestine

Ulf Meyer-Hoffert*,§,1, Mathias Hornef{dagger},{ddagger}, Birgitta Henriques-Normark*,{dagger}, Staffan Normark*, Mats Andersson* and Katrin Pütsep*

* Department of Microbiology Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden;

{dagger} Swedish Institute for Infectious Disease Control, Stockholm, Sweden;

{ddagger} Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany; and

§ Department of Dermatology, University Clinic Schleswig-Holstein, Kiel, Germany

1Correspondence: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 7D-24105 Kiel, Germany. E-mail: umeyerhoffert{at}dermatology.uni-kiel.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The lungs are continuously exposed to a broad array of microbes through inhalation, and microorganisms that escape clearance by the upper airway mucociliary motion will deposit in the alveolar compartment of the lower airways. The pulmonary epithelium in the alveolar compartment is covered by a thin aqueous layer that contains surfactant proteins but also microbicidal components. We have here identified the epithelial cell surface-expressed heparin/heparan sulfate interacting protein (HIP/RPL29) by high-performance liquid chromatography-fractionation, N-terminal sequencing, and mass spectrometry analysis as a major antimicrobial component in extracts of mouse lung tissue. HIP/RPL29 was also detected in extracts of mouse small intestinal tissue. HIP/RPL29 exhibited broad antibacterial activity, notably against Pseudomonas aeruginosa strains. Human recombinant HIP/RPL29 exhibited killing activity in the same order of magnitude. The HIP/RPL29 protein was demonstrated to be localized to the epithelial cells and cell surface of the lungs and intestines by immunohistochemistry. We suggest that HIP/RPL29 fulfills a function as an abundant antibacterial factor of the epithelial innate defense shield against invading bacteria in both the lungs and the small intestine.—Meyer-Hoffert, U., Hornef, M., Henriques-Normark, B., Normark, S., Andersson, M., Pütsep, K. Identification of heparin/heparan sulfate interacting protein as a major broad-spectrum antimicrobial protein in lung and small intestine.


Key Words: epithelial defense • innate immunity • HIP/RPL29 • ribosomal protein • mucosa


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANTIMICROBIAL PEPTIDES ARE KEY effector molecules of the innate immune defense and play a crucial role in protecting the host in a microbially inhabited environment (1) . The majority of antimicrobial peptides exhibit positive net charge and an amphipathic structure, characteristics that confer microbicidal potential (2) . These peptides are produced at body surfaces such as skin, epithelia, and mucosal linings. The reports of peptides and proteins exhibiting antimicrobial activity have increased steadily during the last decade, and the in vivo relevance has been demonstrated in a number of reports (3 4 5 6 7) .

The lungs are exposed continuously to a broad array of microbes through the daily inhalation of thousands of liters of air. Microorganisms that escape clearance by the upper airway mucociliary motion will deposit in the alveolar compartment of the lower airways. The pulmonary epithelium in the alveolar compartment is covered by a thin aqueous layer, the epithelial lining fluid that lines the gas-exchanging epithelium and that contains surfactant proteins but also highly microbicidal components like lysozyme and lactoferrin (8) . These microbicidal components act in concert and, together with resident alveolar macrophages, constitute the immediate defense to avoid tissue damage that otherwise could be the consequence of an inflammatory response. It is only when this immediate defense fails that neutrophils will infiltrate and an inflammatory reaction will be initiated at the alveolar site, including the induction of antimicrobial peptide transcription of the β-defensins (9) .

For both mice and humans, the {alpha}- and the β-defensins constitute the main families of antimicrobial peptides. The {alpha}-defensins are produced at high levels by the Paneth cells of the small intestine in mice, and their role as antimicrobial effector molecules has been clearly demonstrated (5) . Certain bovine epithelial cells are high producers of β-defensins such as tracheal antimicrobial peptide (10) and lingual antimicrobial peptide (11) . In humans and mice, in contrast, only low levels of β-defensins have been demonstrated at various mucosal epithelial surfaces, including the lungs, and mainly at a transcript level. It remains unclear to what extent these β-defensin transcripts are translated into proteins and what the significance of these peptides might be with respect to bactericidal function considering the low expression levels (12) . They may rather have gained other, more cytokine-like functions as alarmins for initiation of the adaptive host defense (13) , and few β-defensins have been demonstrated at microbicidal concentrations in vivo (14) . Because the lungs, in contrast to the small intestine, are not equipped with specialized cells that have the capacity to produce high levels of antimicrobial peptides such as the {alpha}-defensins, we sought to define and isolate major antimicrobial components at a protein level that may be essential for the immediate and constitutive lung innate antibacterial defense.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of lungs, small intestine, and mucus
In a standardized way approved by the local animal legislation, 6- to 8-wk-old C3H/HeN and C57/Bl6 mice were kept under specific pathogen-free conditions. Mice were sacrificed by cervical dislocation following anesthesia. Prior to collection of the lungs, the heart was punctured and the lung vascular system was washed with 0.15 M sodium chloride solution until the lungs turned whitish. The lungs of C57Bl/6 mice and small intestines of C3H/HeN mice were collected and snap-frozen in liquid nitrogen. The frozen tissues were ground in liquid nitrogen, and the powder thus obtained was extracted for 10 min by repeated vortexing with ice-cold 60% aqueous acetonitrile containing 1% trifluoroacetic acid. Following centrifugation at 16,000 g for 20 min, the supernatants were lyophilized and, prior to analysis, dissolved in water with 10% ethanol and cleared by centrifugation. Intestinal mucus was collected by gentle scraping of PBS-washed, longitudinally opened intestine. To assess the purity of the mucus with respect to cellular contamination, the preparation was smeared on a glass slide, air-dried, heat-fixed, and stained with Kwik-Diff stain (Shandon, Pittsburgh, PA, USA).

High performance liquid chromatography (HPLC) purification and analysis of purified components
The extracts were fractionated by reverse-phase HPLC as previously described (15) . The amount of material applied corresponded to 0.25 g of tissue. Elution was performed with 0.18% trifluoroacetic acid in H20 (solvent A) and a gradient of 0.15% trifluoroacetic acid in acetonitrile (solvent B) on a 0.46 x 25 cm Vydac C18 column (The Separation Group, Hesperia, CA, USA) with a flow rate of 0.8 ml/min. For the lungs, initial conditions were 5% solvent B for 10 min, 5–60% solvent B for 110 min, and, finally, 60–95% solvent B for 10 min. For the small intestine extracts, the initial conditions were 5% solvent B for 15 min, 5–52% solvent B for 94 min, and, finally, 52–95% solvent B for 5 min. Fractions of 0.8 ml were collected and lyophilized. For rechromatography, a 0.46 x 15 cm, C18 column ACE 5 µm (ACT, Aberdeen, Scotland) was used with initial conditions of 5% solvent B for 5 min, 5–22% for 8 min, and 22–42% for 40 min. Fractions of 0.4 ml were collected, freeze-dried, and subsequently redissolved in 10 µl of 20% ethanol. N-terminal sequence analysis was done by Edman degradation with a Procise cLC or HT instrument (PE, Applied Biosystems, Foster City, CA, USA). Calculation of peptide amounts was based on equal (80%) coupling efficiency. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis was carried out with a Reflex III (Bruker Daltronics, Bremen, Germany). For these analyses, 1 µl of each fraction was mixed with 1 µl matrix solution ({alpha}-cyano-4 hydroxycinnamic acid or sinnapic acid) in 60% acetonitrile and 0.1% trifluoracetic acid. Spectra were acquired in the linear or the reflectron mode. Calibration was done with external standards.

Proteins and antibodies
Rabbit anti-heparin/heparan sulfate interacting protein (HIP/RPL29) antiserum was raised against a synthetic peptide corresponding to murine HIP/RPL29 amino acids 2–25 (Innovagen, Lund, Sweden). A second polyclonal rabbit anti-HIP/RPL29 antibody (anti-HIP/RPL29 Ab2) raised against a synthetic peptide corresponding to murine HIP/RPL29 amino acids 117–131 has been described previously (16) , and human recombinant HIP/RPL29 was a kind gift of Dr. D. D. Carson (University of Delaware, Newark, DE, USA).

Determination of antibacterial activity
Bacterial strains used were Escherichia coli strain D21; Streptococcus pyogenes clinical isolate; Salmonella enterica; serovar Typhimurium ATCC 14028; Enterococcus faecalis ATCC 29212; Pseudomonas aeruginosa strains PAO1, CF-1, and CF-2; and Listeria monocytogenes type 1 clinical isolate. A low-salt agarose antibacterial assay was used as described (17) , and the zone of inhibited bacterial growth was recorded. For investigation of human recombinant HIP/RPL29, the number of viable (colony-forming) bacteria was analyzed after exposure to rhHIP/RPL29. S. pyogenes or E. coli D21 grown to midlogarithmic phase and 1.25 x 103 bacterial cells were coincubated in 100 µl of sodium phosphate pH 7.2 buffer supplemented with 1% of tryptic soy broth medium with 0.5% yeast extract with or without the addition of peptide (0.1–5 nM). Aliquots were removed after 1 h and plated for counting of the number of viable colony-forming bacteria.

Immunoblotting
Protein extracts and HPLC fractions were separated on a 1.0 mm 10% or 4–12% NuPAGE Bis-Tris Gel (Invitrogen, San Diego, CA, USA) in NuPAGE MES SDS running buffer (Invitrogen). Samples were mixed with NuPAGE LDS sample buffer (Invitrogen), adjusted to 10% β-mercaptoethanol, and heated before loading on the gel and then blotted onto polyvinylidene difluoride filters (Invitrogen). For immuno-dot-blot experiments, 0.5 µl from each lyophilized and redissolved fraction was transferred onto nitrocellulose filters (Hybond-C, Amersham Biosciences, Uppsala, Sweden). The filters were air-dried at room temperature prior to blocking. Filters were blocked in PBS containing 1% Tween (PBST) supplemented with ECL advance blocking reagent, (Amersham Pharmacia Biotech, Uppsala, Sweden). Incubations with antigen-specific antibodies or detecting horseradish peroxidase-conjugated antibodies, respectively, were in PBST. The antibody reactivity was detected by chemiluminescence with SuperSignal West Dura (Pierce Biotechnology, Rockford, IL, USA) alternatively ECL Advance. Western blots were evaluated by densitometry using Intelligent DarkBoxII (FujiFilm, Tokyo, Japan).

Blocking of antibacterial activity
Native HIP/RPL29 purified from murine lungs (n=6) was coincubated with equal protein concentration of whole anti-HIP/RPL29 antiserum and whole preimmunization serum (both diluted ~1:10 in water) as well as anti-HIP/RPL29 Ab2 compared with a similar amount of water. Coincubated HIP/RPL29 was subsequently tested to kill S. pyogenes using the plate diffusion assay as described above.

Immunohistochemistry
Cryosections of healthy C57/BL6 mouse lung intestine were acetone-fixed, blocked in 10% normal goat serum in 4% BSA/PBS, and incubated with rabbit anti-HIP/RPL29 serum or preimmune serum at 1:100 dilution for 1 h at room temperature. After washing, a biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) in combination with the ABC Elite Kit (Vector, Burlingame, CA, USA) was used for detection. Tissue sections incubated with rabbit preimmunization serum served as controls. Murine lung was fixated in 4% paraformaldehyde. Paraffin sections (5 µm) of the tissue samples were deparaffinized and rehydrated before heat-induced antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0). They were subsequently blocked with normal 0.1% BSA and incubated for 60 min with a 1:200 dilution of antiserum or preimmunserum and further stained as cryosections described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification, isolation, and tissues localization of HIP/RPL29
Epithelial primary cells and cell lines have been reported to secrete antimicrobial peptides such as β-defensins (9) , but the repertoire of antimicrobial components in the whole lung tissue is less well defined. Therefore lungs of mice were isolated, extracted by acetonitrile/trifluoracetic acid, and analyzed for antimicrobial components by RP-HPLC and the resulting fractions assayed for antibacterial activity (Fig. 1 A).


Figure 1
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  		Figure 1. Isolation of HIP/RPL29 from lungs. A) RP-HPLC separation of extract from 1 lung of a C57Bl/6 mouse. Top graph: components eluting between 5 and 95% acetonitrile. Bottom graph: antibacterial activity of the fractions with S. pyogenes as indicator strain. Dotted line = acetonitrile concentration. One of 10 similar experiments is shown. Arrow = fractions with high activity to absorbance ratio. B) Rechromatography of fractions 60–61 min (top graph) and subsequent testing for antimicrobial activity (bottom graph). Fraction 24.5–25 min revealed component with N-terminal sequence AKSKNHTTHNQXRKWHN (residue 4 was a modified amino acid interpreted as a methylated Lys), and database search identified HIP/RPL29 protein. C) Mass analysis of HPLC fraction 24.5–25 min from B. All mass values indicate the average mass of the nonprotonated molecules, m/z, mass/charge. D) Immunoblot of fractions 60–61 min using rabbit anti-HIP/RPL29 antiserum revealing a protein with the apparent molecular mass of HIP/RPL29, which is 24 kDa. MW = molecular mass marker (kDa).

We detected an area with relatively high antibacterial activity and low absorbance levels compared with the other components in the chromatogram, shown by the arrow in Fig. 1A . These features are typical for many antimicrobial peptides, and the fractions in this area were therefore pooled and further purified by a second round of RP-HPLC (Fig. 1B ). Following rechromatography, all activity was concentrated to 1 peak (Fig. 1B ), and the peak component was identified by its N-terminal sequence (AKSKNHTTHNQXRKWH) as HIP/RPL29 (GenBank AF236069), a protein known to be expressed at the surface of various epithelial cells (18) . HIP/RPL29 is also identical to the ribosomal protein L29 (RPL29). The mass was determined by mass spectrometry to be 17,499.6 Da, which corresponds to acetylated full-length HIP/RPL29 (Fig. 1C ). Immunoblotting of the pooled 60–62 min fractions displayed in Fig. 1A using specific anti-HIP/RPL29 antibodies revealed a band at 24 kDa (Fig. 1D ). It is known from previous reports that HIP/RPL29 migrates with an apparent molecular weight of 24 kDa in an SDS-PAGE separation (19) . HIP/RPL29 is a basic protein, as are the majority of antimicrobial peptides. This property affects its electrophoretic behavior, and the calculated isoelectric point (pI) of HIP/RPL29 is 11.66.

We next analyzed the tissue localization by immunohistochemical staining of lung tissue with specific antibodies. The staining revealed HIP/RPL29 protein at lung epithelia in the alveolar cells and macrophages but also on the cell surfaces (Fig. 2 ).


Figure 2
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  		Figure 2. Immunohistochemical localization of HIP/RPL29 in lung tissues. Immunohistochemical staining of C57Bl/6 mouse lungs using rabbit anti-HIP/RPL29 antiserum (A) and incubation with preimmunization rabbit serum as a negative control (B). Images x1000 (A) and x100 (B).

Identification of HIP/RPL29 as a major antibacterial factor in the mouse small intestine
We have earlier characterized and isolated a range of antimicrobial components from the small intestine of mice (15 , 17) . On dot-blot analysis of HPLC-fractionated extracts of the small intestine, we detected HIP/RPL29 immunoreactivity in fractions eluting at the same acetonitrile concentration as HIP/RPL29 from the lungs. These fractions exhibited antibacterial activity of the same magnitude as those fractions that contained cryptdins and/or cryptdin-related sequence (CRS) peptides (17) (Fig. 3 A). Immunoblot analysis displayed a band at 24 kDa in the fraction with anti-HIP/RPL29 reactivity. The mucus of the small intestine, which had been gently scraped from the underlying tissue and was essentially cell free, exhibited anti-HIP/RPL29 reactivity, thus indicating extracellular localization (Fig. 3B ). Further immunohistochemical staining for detecting tissue localization revealed a highly selective expression of HIP/RPL29 protein mainly at the epithelial lining and most evident at the bottom of the crypts where the Paneth cells are located (Fig. 4 ).


Figure 3
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  		Figure 3. Identification of HIP/RPL29 in the small intestine and small intestinal mucus. A) RP-HPLC separation of extract from small intestine of a C3H/HeN mouse. Fractions eluting between 50 and 75 min were collected (top graph) and evaluated by analysis of antibacterial activity (bottom graph). Brackets = known regions of antimicrobial {alpha}-defensins and CRS peptides. Detection of HIP/RPL29 by immunodot-blot is displayed as insets onto the fractions exhibiting antibacterial activity. B) Left panel: HIP/RPL29 detection using immunoblotting of the pooled fractions eluting at 68–69 min in A. Right panel: immunoblot of extracts from total small intestine and small intestine mucus preparation, respectively. MW = molecular mass marker (kDa).


Figure 4
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  		Figure 4. Immunohistochemical staining of HIP/RPL29 in the intestine. Immunohistochemical staining of C3H/HeN mouse small intestine using anti-HIP/RPL29 antiserum (A, B) and preimmunization serum as negative control (C). Strong immunoreactivity is detected predominantly at the epithelial lining and at the bottom of the crypts (arrows).

Antibacterial activity of HIP/RPL29
To certify that the antibacterial activity of the isolated HIP/RPL29 was from the HIP/RPL29 protein and not from contaminating substances, an experiment was designed to test whether anti-HIP/RPL29 antibodies against the isolated HIP/RPL29 protein could abrogate the bactericidal activity. Indeed, the anti-HIP/RPL29 antiserum raised against the N-terminal part of HIP/RPL29 inhibited the antibacterial activity of the isolated HIP/RPL29 completely, whereas a preimmunization serum control had no effect (Fig. 5 A). The second antibody raised against a synthetic peptide corresponding to murine HIP/RPL29 amino acids 117–131 diminished the activity by ~60% (Fig. 5A ). These results confirmed that the antibacterial activity detected was from the HIP/RPL29 protein.


Figure 5
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  		Figure 5. Bactericidal repertoire of HIP/RPL29. A) Inhibition of bactericidal activity against S. pyogenes by coincubation of the HIP/RPL29 containing pooled HPLC fractions (60–62 min in Fig. 1A ) with anti-HIP/RPL29 antiserum, a second anti-HIP/RPL29 antibody (Ab2), as well as the preimmunization serum. Killing of bacteria was tested using the diffusion assay. Antibacterial activity of 1 µl of fraction 60–62 min was set as 100%; bars = mean (n=3); error bars = SD. B) Antibacterial activity of HIP/RPL29 against various gram positive and gram negative bacteria against purified mHIP/RPL29 using the plate diffusion assay. The killing zone diameter was measured and given as antibacterial activity (mm). Bars = 1 result of 2 similar experiments. C) Extracts of murine lungs were incubated with anti-HIP/RPL29 serum and tested to kill S. pyogenes compared with lung extracts incubated only with preimmunization serum (n=8; P<0.05; unpaired Student’s t test). The total antibacterial activity of HIP/RPL29 was ~5% of total lung extracts.

To evaluate the impact of HIP/RPL29 on the total antibacterial activity of lung extracts, we preincubated the total extract of a single mouse lung either with anti-HIP/RPL29 antiserum or preimmunization serum (10:1; v/v) and subsequently added the mixture to log-phase growing S. pyogenes. Extracts of lungs incubated with anti-HIP/RPL29 serum were significantly less efficient to kill S. pyogenes than were lung extracts incubated only with preimmunization serum (n=8, P<0.05 applying unpaired Student’s t test). The total antibacterial activity of HIP/RPL29 was ~5% of total lung extracts (Fig. 5C ).

Next, purified HIP/RPL29 was tested for bactericidal function against different lung- and intestinal bacterial pathogens. HIP/RPL29 exhibited activity against the gram positive bacteria L. monocytogenes, S. pyogenes and the gram negative bacteria, E. coli and different strains of P. aeruginosa (Fig. 5B ). Interestingly, growth of Salmonella typhimurium was not affected by HIP/RPL29. Because murine and human HIP/RPL29 differ slightly in the C-terminal protein sequence, we assessed human recombinant HIP/RPL29 for antibacterial activity using the growth assay in liquid media. Notably, human recombinant HIP/RPL29 was active against E. coli and S. pyogenes in nanomolar range (Fig. 6 ).


Figure 6
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  		Figure 6. Antibacterial activity of human recombinant HIP/RPL29. Human recombinant HIP/RPL29 was tested against S. pyogenes and E. coli using a liquid bacterial growth assay, and surviving bacteria are given as colony-forming units (CFU). Squares = mean (n=3); error bars = SD; *P < 0.05; Student’s t test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we aimed to identify major substances that might contribute to the antimicrobial shield of the epithelial surface in the lungs. Using a similar strategy, which has been successful to purify {alpha}-defensins and CRS peptides from the small intestine of single mice (15 , 17) , we identified HIP/RPL29 as one of the more abundant antimicrobial components in the extracts of the lungs of individual mice. It represents the first cationic antimicrobial component to be purified from murine lungs, to our knowledge. It exhibited broad antibacterial activity against gram negative as well as gram positive bacteria and localized to the surface of the epithelial cells. HIP/RPL29 contributed to ~5% of the total killing activity of lung extracts, which can be considered rather high when taking into account the complex mixture of components.

Moreover, HIP/RPL29 was also identified in the extracts of the small intestine of mice. Its activity was in the same range as the activity of the previously described cryptdins and CRS peptides (17 , 20) . Interestingly, HIP/RPL29 expression localized to epithelial cells and predominantly to the crypts, where Paneth cells are present and produce most of the known antimicrobial components of the small intestine. In addition, HIP/RPL29 was detected in the overlying mucus. The mechanism of how HIP/RPL29 localizes to the mucus is unclear. It may be that HIP/RPL29 derives from shredded epithelial cells during the process of epithelial regeneration or that it is secreted from the crypt epithelial cells, including the Paneth cells at the bottom of the crypts, similar to other antimicrobial components.

HIP/RPL29 was first described as a highly basic heparin/heparin sulfate-interacting protein in uterine endometrium with strong cell surface expression in luminal uterine epithelial cells (18) . Significant expression of HIP/RPL29 was reported in murine embryonic tissues, notably the eye, liver, lung, and cartilage of the developing bones (21) and predominantly localized to epithelial cells. By homology search, it was found that HIP/RPL29 is identical to the cDNA sequence of ribosomal protein L29 (22) . Although the exact in vivo function of HIP/RPL29 is not known, remarkably diverse biological properties have been attributed to HIP/RPL29. It is suggestive that HIP/RPL29 is not produced to the same extent and same tissues in adult individuals as during fetal life, supporting the idea that HIP/RPL29 function is not associated exclusively with protein synthesis. It is not unusual for ribosomal proteins to have an extraribosomal function (23 24 25) , and the presence of HIP/RPL29 at the cellular surface is not unique because the ribosomal protein (LBP/p40/RPSa) also was found to be exposed at the cell surface (26) . Notably, knockout mice lacking HIP/RPL29 are viable but exhibit growth deficiencies during fetal development (27) . The idea that HIP/RPL29 plays more than one role in cellular biology is supported by described in vivo and in vitro changes in the cytological distribution of HIP/RPL29 during mouse mammary epithelial cell expression and differentiation (28) .

HIP/RPL29 is found tightly associated with membrane fractions (19 , 29) yet predicted amino acid sequence analysis of either human or mouse HIP/RPL29 cDNA does not indicate the presence of an obvious N-terminal secretion signal sequence or transmembrane segment. HIP/RPL29 association with these membranes is disrupted by high salt concentration but not by heparin or heparinase treatment, suggesting attachment of HIP/RPL29 other than heparan sulfate proteoglycans (19 , 30 31 32) .

Our finding that HIP/RPL29 exhibits broad-spectrum antibacterial activity and taking into account its cellular expression and localization, it appears likely that HIP/RPL29 acts as an antimicrobial factor at the epithelial lining throughout the body-protecting cell surfaces against invading bacteria but also balancing the normal flora. As an antimicrobial component in the intestinal mucus, HIP/RPL29 contributes to a chemical barrier to separate the intestinal epithelial lining from the intestinal flora (33) .

Helicobacter pylori grown in vitro displayed antibacterial activity that could be traced to several cecropin-like peptides derived from the N-terminal part of ribosomal protein L1 (34) . Ribosomal proteins have an affinity for negatively charged molecules and were identified as antimicrobial agents in the skin mucus of fish (35) , human colon epithelium (specifically, RPL30, RPL39, and RPS19) (36) , and human colon (37) . Ubiquicidin, which is a bactericidal protein that can be isolated from the cytoplasm of {gamma}-interferon-activated macrophages, is identical to RPS30 (38) . It is well known that histones from a variety of species, including humans and mice, may exert strong microbicidal activity (37 , 39 40 41 42) . These peptides/polypeptides are stored in the cytoplasm and many have been shown to be secreted (42 , 43) . Furthermore, histones have been demonstrated to decorate extracellular traps produced by neutrophils (44) , which kill trapped bacteria. In contrast to some histones that are cleaved to generate the antimicrobial capacity (41 , 43) , the entire molecule of HIP/RPL29 is antibacterial. This is similar to larger antibacterial proteins such as NK-lysin or attacin (45 , 46) , demonstrating that small size is not a prerequisite for antibacterial effect. It seems that motifs with basic amino acids of certain ribosomal proteins and of histones have functions as innate antimicrobial defense molecules because of to their affinity for negatively charged molecules and, hence, microbial surfaces.

In summary, we have identified HIP/RPL29 as an abundant antimicrobial protein in the lungs with broad antimicrobial activity, including the pathogen P. aeroginosa, which confers stable constitutive protection of the alveolar cell compartment and requires no specific induction. The same protein, HIP/RPL29, localizes also to the mucus of the small intestine of mice and specifically to the crypt Paneth cell region. Recently, it has been demonstrated that the stem cells of the small intestine are located intermingled between the Paneth cells (47) at the bottom of the crypts. An antibacterial component such as HIP/RPL29 that requires no prior induction may play an important function in the protection of the crypt stem cells against potentially harmful bacteria.


   ACKNOWLEDGMENTS
 
We thank E. Cederlund for performing the Edman degradation and D. Gütle for excellent technical work. This work was supported by grants from the German Research Foundation to U.M.H. (Me2037/2-1) and to M.H. (HO 2236/5-2); the Swedish Research Council to K.P., M.H., M.A., and B.H.N.; Ruth och Richard Juhlins Stiftelse to K.P.; Cancerfonden and Hannover Medical School to M.H.; the Scandinavian Society for Antimicrobial Chemotherapy and the Swedish Foundation for International Cooperation in Research and Higher Education to M.A.; the Swedish Royal Academy of Sciences, Torsten and Ragnar Söderbergs Foundation, and Swedish Foundation for Strategic Research to B.H.N.; and Thyssen Foundation to M.H. (AZ 10.05.2.173).

Received for publication December 3, 2007. Accepted for publication February 7, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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